Methods and compositions for preventing and treating auditory dysfunctions

ABSTRACT

The invention provides methods for treating auditory impairments in a subject in need of treatment comprising administering to said subject an effective amount of a composition comprising, as an active agent, one or more of a carboxy alkyl ester, a quinic acid derivative, a caffeic acid derivative, a ferulic acid derivative, or a quinic acid lactone or derivative thereof or pharmaceutically acceptable salt thereof and an acceptable carrier or excipient, so as to treat auditory impairments in the subject.

This patent application claims the benefit of the filing date of U.S.Ser. No. 61/653,577, filed May 31, 2012 and U.S. Ser. No. 61/701,397,filed Sep. 14, 2012, the contents of all of which are hereinincorporated by reference in their entireties into the present patentapplication.

Throughout this application various publications are referenced. Thedisclosures of these publications in their entireties are herebyincorporated by reference into this application in order to more fullydescribe the state of the art to which this invention pertains.

FIELD OF THE INVENTION

This invention relates to auditory dysfunctions (e.g., hearing loss,tinnitus, hyperacusis, and related auditory processing disorders) andmethods of preventing and treating such dysfunctions. Additionally, thisinvention relates to specific chemicals and chemical compositions andthe uses of such chemicals and chemical compositions for preventingand/or treating auditory dysfunctions.

BACKGROUND OF THE INVENTION

Auditory dysfunction in people is an ongoing problem in the medicalfields of otology and audiology. Auditory dysfunctions typically arisefrom both acute and chronic exposures to loud sounds, ototoxic chemicalsand aging. Sounds exceeding 85 decibels can cause hearing loss and isgenerated by sound sources such as, gun shots, exploding bombs, jetengines, power tools, and musical concerts. Other common everydayactivities and products also give rise to high intensity noise such asuse of hair dryers, MP3 players, lawn mowers, and blenders. Militarypersonnel are particularly at risk for noise induced hearing loss due totypical military noise exposures. Side effects to noise induced hearingloss include tinnitus (ringing in the ears), diminished speechunderstanding, hyperacusis, recruitment and various types of auditoryprocessing impairments. Exposures to commonly used medications may alsoinduce auditory dysfunctions. For instance, patients treated withanticancer therapies, antibiotics and other medications often develophearing loss as a side effect. Furthermore, exposure to industrialchemicals and gasses may induce auditory impairments. Lastly, auditorydysfunction is a common consequence of aging in Western societies.

Approximately 17 percent of Americans (estimated at 36 million) havehearing loss and half of that number are under the age of 65. It ispredicted that the number of Americans with hearing loss will exceed 70million by the year 2030.

About 300 million people worldwide currently suffer from moderate tosevere hearing loss, and this number is expected to increase to 700million by the year 2015. Most of these people will suffer from noiseinduced hearing loss and one in four Americans will develop permanenthearing loss as a result of occupational exposure to noise hazards.According to the Center for Commercialization of Advanced Technology,the Department of Defense and the VA, the VA spends over $1 billion onhearing loss compensation. The Navy, Marine Corps, and Air Force(combined) file 22,000 new hearing loss claims, and hearing loss coststhe economy more than $56 billion per year.

Very few cases of hearing loss can actually be cured. Audiologicaldevices such as hearing aids have limitations including the inability toimprove speech intelligibility. Of those impacted by hearingimpairments, less than 20 percent presently use hearing instruments.

In cases of age-related, noise- or drug-induced auditory dysfunctions,the only effective way to currently “treat” the disorder or reduce itsseverity is prevention: avoiding excessive noise and using earprotectors, practicing a healthy lifestyle, and avoiding exposure toototoxic drugs and substances if possible.

Once the hearing loss has developed, people may use a hearing aid tocorrect the inability to hear. However, despite advances in theperformance of these prostheses, they still have their limits. Forexample, hearing aids mainly amplify sound and cannot correct forsuprathreshold or retrocochlear impairments such as impaired speechintelligibility, speech in noise deficits, tinnitus, hyperacusis,loudness recruitment and various other types of central auditoryprocessing disorders. Hearing aids essentially amplify sounds whichstimulate unimpaired cells but there is no therapy for aiding recoveryof impaired cells or maximizing the function of existing unimpairedcells. In cases of complete or profound deafness, a cochlear implant maybe used. This device transmits electrical stimuli via electrodessurgically implanted into the cochlea. A cochlear implant can be ofparticular help for deaf children if it is implanted around the age oftwo or three, the time when language skills are developing fastest.However, cochlear implants involve invasive surgery and are expensive.Furthermore, cochlear implants require viable neurons in order toachieve benefit.

Thus, there remains a long felt need to protect auditory cells beforeinjury and preserve/promote the function of existing cells after injury.As disclosed below, the present invention provides a novel means forpreventing and treating auditory dysfunctions.

SUMMARY OF THE INVENTION

The invention provides methods for treating auditory impairments in asubject in need of treatment comprising administering to said subject aneffective amount of a composition comprising, as an active agent, one ormore of a carboxy alkyl ester, a quinic acid derivative, a caffeic acidderivative, a ferulic acid derivative, or a quinic acid lactone orderivative thereof or pharmaceutically acceptable salt thereof and anacceptable carrier or excipient, so as to treat auditory impairments inthe subject.

In one embodiment, the invention provides methods for treating auditoryimpairments in a subject comprising administering to said subject aneffective amount of a composition comprising as an active agent one ormore of a carboxy alkyl ester, alkaloid, pentacyclic alkaloid, tannin,or phytochemical derived from the inner bark or root of Uncariatomentosa or derivative thereof or pharmaceutically acceptable saltthereof and an acceptable carrier or excipient, so as to treat auditoryimpairments in the subject.

In another embodiment, the invention provides methods for inhibitingcochlear inflammation in a subject comprising administering to saidsubject an effective amount of a composition comprising as an activeagent one or more of a carboxy alkyl ester, a quinic acid derivative, acaffeic acid derivative, a ferulic acid derivative, or a quinic acidlactone or derivative thereof or pharmaceutically acceptable saltthereof and an acceptable carrier or excipient, thereby inhibitingcochlear inflammation in the subject.

In another embodiment, the invention provides methods for inhibitingcochlear inflammation in a subject comprising administering to saidsubject an effective amount of a composition comprising as an activeagent one or more of a carboxy alkyl ester, alkaloid, pentacyclicalkaloid, tannin, or phytochemical derived from the inner bark or rootof Uncaria tomentosa or derivative thereof or pharmaceuticallyacceptable salt thereof and an acceptable carrier or excipient, therebyinhibiting cochlear inflammation in the subject.

In one embodiment, the invention provides for methods for inhibiting theloss or death of the cells of the auditory system in a subjectcomprising administering to said subject an effective amount of acomposition comprising as an active agent one or more of a carboxy alkylester, a quinic acid derivative, a caffeic acid derivative, a ferulicacid derivative, or a quinic acid lactone or derivative thereof orpharmaceutically acceptable salt thereof and an acceptable carrier orexcipient, thereby inhibiting loss or death of the cells of the auditorysystem in the subject.

In another embodiment, the invention provides methods for inhibiting theloss or death of the cells of the auditory system in a subjectcomprising administering to said subject an effective amount of acomposition comprising as an active agent one or more of a carboxy alkylester, alkaloid, pentacyclic alkaloid, tannin, or phytochemical derivedfrom the inner bark or root of Uncaria tomentosa or derivative thereofor pharmaceutically acceptable salt thereof and an acceptable carrier orexcipient, thereby inhibiting loss or death of the cells of the auditorysystem in the subject.

In a further embodiment, the invention provides methods for maintainingor promoting the growth of cells of the auditory system in a subjectcomprising administering to said subject a composition comprising as anactive agent one or more of a carboxy alkyl ester, a quinic acidderivative, a caffeic acid derivative, a ferulic acid derivative, or aquinic acid lactone or derivative thereof or pharmaceutically acceptablesalt thereof and an acceptable carrier or excipient in an effectiveamount so as to augment endogenous DNA repair, thereby maintaining orpromoting the growth of cells of the auditory system in the subject.

In another embodiment, the invention provides methods for maintaining orpromoting the growth of cells of the auditory system in a subjectcomprising administering to said subject a composition comprising as anactive agent one or more of a carboxy alkyl ester, alkaloid, pentacyclicalkaloid, tannin, or phytochemical derived from the inner bark or rootof Uncaria tomentosa or derivative thereof or pharmaceuticallyacceptable salt thereof and an acceptable carrier or excipient in aneffective amount so as to augment endogenous DNA repair, therebymaintaining or promoting the growth of cells of the auditory system inthe subject.

In another embodiment, the invention provides methods for inhibiting orreversing ion dyshomeostasis, mitochondriopathy, energy catastropheand/or the proliferation of free radicals in the auditory system in asubject comprising administering to said subject an effective amount ofa composition comprising as an active agent one or more of a carboxyalkyl ester, a quinic acid derivative, a caffeic acid derivative, aferulic acid derivative, or a quinic acid lactone or derivative thereofor pharmaceutically acceptable salt thereof and an acceptable carrier orexcipient in an effective amount so as to maintain the viability orgrowth of auditory nerve cells or cochlear cells, thereby inhibiting orreversing ion dyshomeostasis, mitochondriopathy, energy catastropheand/or the proliferation of free radicals in the auditory system.

In another embodiment, the invention provides methods for inhibiting orreversing ion dyshomeostasis, mitochondriopathy, energy catastropheand/or the proliferation of free radicals in the auditory system in asubject comprising administering to said subject an effective amount ofa composition comprising as an active agent one or more of a carboxyalkyl ester, alkaloid, pentacyclic alkaloid, tannin, or phytochemicalderived from the inner bark or root of Uncaria tomentosa or derivativethereof or pharmaceutically acceptable salt thereof and an acceptablecarrier or excipient in an effective amount so as to maintain theviability or growth of auditory nerve cells or cochlear cells, therebyinhibiting or reversing ion dyshomeostasis, mitochondriopathy, energycatastrophe and/or the proliferation of free radicals in the auditorysystem.

The invention also provides pharmaceutical formulations comprising anyone or more of a carboxy alkyl ester, a quinic acid derivative, acaffeic acid derivative, a ferulic acid derivative, or a quinic acidlactone or a derivative thereof or pharmaceutically acceptable salt andpharmaceutically acceptable excipients.

The invention also provides pharmaceutical formulations comprising anyone or more of a carboxy alkyl ester, pentacyclic alkaloid, tannin, orphytochemical derived from the inner bark or root of Uncaria tomentosaor a derivative thereof or pharmaceutically acceptable salt thereof andpharmaceutically acceptable excipients.

BRIEF DESCRIPTION OF THE FIGURES

The figures show that gastric gavage of laboratory rats with aformulation of the agents result in almost complete recovery from noiseinduced hearing loss. The noise induced hearing loss was induced with adamaging noise dose that exceeds the permissible doses for work-placesafety in the United States. Note that both sensory and neural functionswere preserved due to treatment with the agent. Additionally, the numberof dead/missing auditory cells was reduced in the group that was treatedwith the agent and the noise while the group that received only thenoise dose showed significant cellular loss.

FIG. 1. Sensory Function. DPOAE levels as a function of ƒ₂ frequency areshown for each treatment group: (A) control (water gavage-only), (B)noise exposure-only (no gavage), (C) CAE-only and (D) CAE+noise. Thegray bars in this and subsequent figures represent the frequency rangeof the damaging noise. Note that the noise-only group showed depressedDPOAE levels out to 4 weeks after noise exposure while the CAE+noisegroup showed almost complete recovery as early as 1 week following thenoise exposure. Errors bars are standard errors of the means.

FIG. 2. Cytocochleograms of the peripheral auditory neurosensoryepithelium. Photomicrograph of the peripheral auditory neurosensoryepithelium is shown in (A) with dark arrows pointing to missing outerhair cells (OHCs) in row 1 of rows 1-3 of OHCs. OHC counts (calledcytocochleograms) are displayed for (B) control (water gavage-only), (C)noise exposure-only (no gavage), (D) CAE-only (no noise exposure) and(E) CAE+noise treated groups. These cytocochleograms reveal the percentof missing OHCs from rows 1-3 as a function of distance (0-9 mm) andfrequency (1-50 kHz). Note that the noise exposure produced OHCs loss,however the level of missing OHCs is less in the CAE+noise groupcompared to the noise-only group. Errors bars are standard errors of themeans.

FIG. 3. Neural Function. CAP recordings of neural sensitivity in dB SPLare shown for each group. Note that the CAE+noise group exhibit betterneural sensitivity than the noise-only group. Errors bars are standarderrors of the means.

FIG. 4. Chemical Structure. The structure of three representativecarboxy alkyl esters are shown: (1) 3,4-O-dicaffeoylquinic acid; (2)3-O-feruloylquinic acid; (3) 3-O-caffeoylquinic acid.

FIG. 5. Positive and negative controls. (A) The immunohistochemistryprocedure produced prominent reaction products (see arrows) withinhepatocytes (positive control cells). (B-D) Omitting the primaryantibody from the immunohistochemistry procedure resulted in negativereaction products (negative control). The respective blocking serum foreach antibody is indicated. The abbreviation CV is central vein. Scalesbar (10 μm) in (A) applies to all panels.

FIG. 6. Representative examples of the intracellular distributionpatterns. (A) Photomicrograph of a field of neurons exhibiting diffuseexpression. (B) Enlargement of the area outlined in (A) showing thatreaction products were distributed throughout the soma. (C) Arepresentative 1-pixel wide linescan demonstrate that neurons with thisdiffuse pattern exhibit a specific morphologic profile where chromogenintensity is linear across the soma. The y-axis in panels C, F, I and Lare inverted gray (g) levels (1/g). (D) Photomicrograph of a field ofneurons exhibiting cytoplasmic expression. (E) Enlargement of the areaoutlined in (D) showing that reaction products were predominantlylocalized in the cytoplasm. (F) A representative 1-pixel wide linescandemonstrate that cytoplasmic reactive neurons exhibit a specificmorphologic profile where chromogen intensity in the nucleoplasm isminimal compared to the cytoplasm. (G) Photomicrograph of a field ofneurons exhibiting nuclear and diffuse expression. (H) Enlargement ofthe area outlined in (G) showing nuclear reactive neurons. (I) Arepresentative 1-pixel wide linescan reveal that nuclear reactiveneurons exhibit a specific morphologic profile where chromogen intensityin the nucleoplasm is maximal compared to the cytoplasm. (J)Photomicrograph of a field of neurons exhibiting perinuclear expression.(K) Enlargement of the area outlined in (J) showing reaction productswere predominantly localized around the nucleus with residual stainingaround the plasmalemma. (L) A representative 1-pixel wide linescanreveal that the perinuclear localization pattern exhibits a specificmorphologic profile where chromogen intensity is maximal at the nuclearannulus. The scale bar (20 μm) in panel A applied to panels D, G and J.The scale bar (10 μm) in panel B applied to panels E, H, and K.

FIG. 7. Allocation of repair proteins across subcellular compartments.The panels illustrate cell counts from the control group (normal, N=5).Each subcellular compartment is enriched with at least one repairprotein. For instance, the XPC protein is predominantly localized in thecytoplasm while XPA is primarily diffused throughout the cytoplasm andnucleus. Additionally, the CSA protein exhibits a preference forcytoplasmic and perinuclear loci. Each bar represents mean±SEM.

FIG. 8. Re-allocation of repair proteins to the cytoplasmic compartment.The panels illustrate cell counts from the noise exposed group (N=5).Note that all the proteins exhibited preferential localization in thecytoplasm. Each bar represents mean±SEM.

FIG. 9. Allocation of repair proteins across subcellular compartments.The panels illustrate cell counts from the CAE treated group (N=3). TheCAE treatment apparently equalized the distribution of the proteinsacross patterns. For instance, statistical analyses reveal that no onelocalization pattern is significantly different than the other patternsfor a given protein. Each bar represents mean±SEM.

FIG. 10. Mixed allocation of repair proteins across subcellularcompartments. The panels illustrate cell counts from the CAE+noisetreated group (N=3). CAE+noise equalized the intracellular distributionof the XPC and XPA proteins. For instance, statistical analyses revealthat no one localization pattern is significantly different than theother patterns. However, the CSA protein exhibited a preference forcytoplasmic and perinuclear loci. Each bar represents mean±SEM.

FIG. 11. The effect of the experimental conditions on the distributionpatterns. The experimental conditions did not significantly change thedistribution patterns for the XPC protein. However, there weresignificant changes for the XPA and CSA proteins. The experimentalconditions were control (N=5), noise (N=5), CAE (N=3) and CAE+noise(N=3). Each plot displays the mean±SEM. Statistical analyses includingpot-hoc test results are described in the text.

FIG. 12. γ-H2Ax labeling in kidney. Panel A shows γ-H2Ax immunolabelingin the Long-Evans rat kidney (positive control). A few podocytes (P)that line the urinary space (US) are labeled while other podocytes arenot labeled. The tubular epithelia (TE) in panel A shows little or nostaining Panel B reveals that omitting the antibody from theimmunolabeling procedure resulted in no staining within podocytes(negative control). The scale bar (20 μm) in panel B applies to panel A.

FIG. 13. γ-H2Ax labeling in scala media. Panel A shows that severalstructures are labeled but the most prominent is the organ of Corti.γ-H2Ax labeling can be detected in the organ of Corti with (panel C) andwithout (panel B) noise exposure. The labeling is predominantlylocalized in hair cells and supporting cells. Abbreviations: Oc, organof Corti; Ohc, outer hair cell; Ihc, inner hair cell. The scale bar inpanel A is 100 μm and the scale bars in panels B and C are 10 μm.

FIG. 14. γ-H2Ax levels within the organ of Corti. γ-H2Ax levels werequantified in mathematical energy units per pixel (E_(m)/pix) anddisplayed in panels A-B as a function of the treatment conditions. Notethat there were no significant differences in γ-H2Ax level between thecontrol and CAE conditions. However, there was a marked increase afternoise or co-treatment with CAE and noise. The level of γ-H2Ax issignificantly lower in the CAE+noise condition relative to thenoise-only condition. This effect is further supported in Panel B whereγ-H2Ax levels for individual cochlear turns were quantified. Errors barsare standard errors of the means.

FIG. 15. Protection from noise injury. The levels of 2ƒ₁-ƒ₂ as afunction of ƒ₂ frequency (DP-gram; L₁/L₂=65/55) are shown for eachtreatment group at baseline and 1 day post-noise exposure. The verticalgray bar in this and all figures represent the frequency range of thedamaging noise. Both the noise-only group and the CAE+noise group wereexposed in the same noise exposure chamber at the same time. Note thatthe CAE+noise group exhibited better (higher) levels than the noise onlygroup at 1 day post-noise exposure. Errors bars are standard errors ofthe means.

FIG. 16. Recovery from noise injury. DP-grams (L₁/L₂=55/35) obtained at1 day, 1 week and 4 weeks post noise exposure are illustrated for thecontrol, noise and CAE+noise groups. Both the noise-only group and theCAE+noise group were exposed in the same noise exposure chamber at thesame time. Note that the CAE treated group showed faster recovery thanthe noise exposure group. The continuous gray lines in each panelrepresent baseline recordings and the broken gray lines are the noisefloor. Errors bars are standard errors of the means.

FIG. 17. Preliminary data showing that seven days of CAE treatmentstarting at 1 day after noise exposure resulted in accelerated recoveryof auditory sensitivity (threshold) as determined by auditory brainstemresponse (ABR). Panel A (short term study; CAE 160 mg/kg/7 days) revealsthat both the noise and CAE+noise groups exhibited the same level ofloss at 1 day after the noise injury however, the CAE+noise groupsshowed significantly (ANOVA main effects of groups: p<0.05) fasterrecover at just 1 week after the noise exposure. Panel B (long termstudy; CAE 160 mg/kg/28 days) shows frequency specific thresholds at 1month after the noise exposure. Note that the thresholds for theCAE+noise group is significantly (ANOVA main effects of groups: p<0.05)better (lower) than that of the noise-only group. Panel C (long termstudy; CAE 160 mg/kg/28 days) shows that at 1 month after noiseexposure, the threshold shift (ΔΦ: relative to baseline) for theCAE+noise group is not significantly different (ns) from that ofcontrol. However, the noise-only group exhibited significant thresholdshift compared to that of control. Each plot/bar represents mean±SEM forN=6 or 8 rats.

DETAILED DESCRIPTION OF THE INVENTION

Definitions

Before describing the invention in detail, several terms used in thecontext of the present invention will be defined. In addition to theseterms, others are defined elsewhere in the specification as necessary.Unless otherwise expressly defined herein, terms of art used in thisspecification will have their art-recognized meanings.

Other features and advantages of the invention will be apparent from thefollowing drawings, detailed description, and appended claims.

An “agent” or “active agent” refers to an active ingredient delivered toachieve an intended protective or therapeutic benefit. As used herein,an active agent can be any one or more of the following chemicals;phenylpropanoids, carboxy alkyl esters, alkaloids, pentacyclicalkaloids, tannins, phytochemicals, chlorogenic acids, quinic acidesters and quinic acids, and derivatives thereof and pharmaceuticallyacceptable salts thereof.

The term “combination therapy” refers to a therapeutic regimen thatinvolves at least two of the agents. For example, a combination therapymay involve the administration of a chlorogenic acid and a quinic acid.Alternatively, a combination therapy may involve the administration ofone or more agent in conjunction with another therapeutic chemical,compound or device. In the context of the administration of two or morechemically distinct agents, it is understood that the active ingredientsmay be administered as part of the same composition or as differentcompositions. When administered as separate compositions, thecompositions comprising the different active ingredients may beadministered at the same or different times, by the same or differentroutes, using the same or different dosing regimens, all as theparticular context requires. Similarly, when one or more agents arecombined with other drugs, the drug(s) may be delivered before, during,and/or after the period the subject or person is in therapy.

In the context of this invention, a “liquid composition” refers to onethat, in its filled and finished form as provided from a manufacturer toan end user (e.g., a doctor, nurse, or patient), is a liquid orsolution, as opposed to a solid. Here, “solid” refers to compositionsthat are not liquids or solutions. For example, such solids includedried compositions prepared by filtering, lyophilization, freeze-drying,precipitation, drying and similar procedures.

“Monotherapy” refers to a treatment regimen based on the delivery of onetherapeutically effective composition of the gents, whether administeredas a single dose or several doses over time.

A “plurality” means more than one.

The term “species” when used in the context of describing a particulardrug species, refers to a population of chemically indistinct molecules.

A “subject” or “patient” refers to an animal in need of treatment thatcan be effected by molecules of the invention. Animals that can betreated in accordance with the invention include vertebrates, withmammals such as bovine, canine, equine, feline, ovine, porcine, murineand primate (including humans and non-human primates) animals beingparticularly preferred examples. Subjects in need of treatment includethose with auditory dysfunctions (e.g., hearing loss, tinnitus,hyperacusis, and related auditory processing disorders) and subject thathave a familial history of hearing impairments or auditory dysfunctions.

DP-grams as referred to herein are graphs of distortion productotoacoustic emissions.

Gavage, as used herein refers to supplying a nutritional substance intothe stomach.

The term “pharmaceutically acceptable salt” refers to salts which retainthe biological effectiveness and properties of the agents, and which arenot biologically or otherwise undesirable. In many cases, the agents ofthis invention are capable of forming acid and/or base salts by virtueof the presence of amino and/or carboxyl groups or groups similarthereto. Pharmaceutically acceptable acid addition salts may be preparedfrom inorganic and organic acids, while pharmaceutically acceptable baseaddition salts can be prepared from inorganic and organic bases. For areview of pharmaceutically acceptable salts, see, e.g., Berge, et al.(J. Pharm. Sci., vol. 66, 1 (1977)).

The expression “non-toxic pharmaceutically acceptable salts” refer tonon-toxic salts formed with nontoxic, pharmaceutically acceptableinorganic or organic acids or inorganic or organic bases. For example,the salts include those derived from inorganic acids such ashydrochloric, hydrobromic, sulfuric, sulfamic, phosphoric, nitric, andthe like, as well as salts prepared from organic acids such as acetic,propionic, succinic, glycolic, stearic, lactic, malic, tartaric, citric,ascorbic, palmitic, maleic, hydroxymaleic, phenylacetic, glutamic,benzoic, salicylic, sulfanilic, fumaric, methanesulfonic,trifluoromethanesulfonic, and toluenesulfonic acid and the like. Saltsalso include those from inorganic bases, such as ammonia, sodiumhydroxide, potassium hydroxide, and hydrazine. Suitable organic basesinclude methylamine, ethylamine, propylamine, dimethylamine,diethylamine, diethanolamine, trimethylamine, triethylamine,triethanolamine, ethylenediamine, hydroxyethylamine, morpholine,piperazine, and guanidine, as the case may be as use of such salts areamenable to the agents.

The term “effective amount” of a compound (or composition of theinvention, or the like) means an amount that is effective to exhibit thedesired biological activity or achieve the desired clinical result in asubject response to the particular treatment, commensurate with areasonable benefit/risk ratio when used in the manner of this invention.

A “therapeutically effective amount” refers to an amount of an activeingredient sufficient to effect treatment when administered to a subjectin need of such treatment. In the context of auditory treatment, a“therapeutically effective amount” is one that produces an objectiveresponse in evaluable patients. Such responses include changes in one ormore parameters associated with auditory function and structure. Thetherapeutically effective amount will vary depending upon the particularsubject and condition being treated, the weight and age of the subject,the severity of the disease condition, the particular compound chosen,the dosing regimen to be followed, timing of administration, the mannerof administration and the like, all of which can readily be determinedby one of ordinary skill in the art. It will be appreciated that in thecontext of combination therapy, what constitutes a therapeuticallyeffective amount of a particular active ingredient may differ from whatconstitutes a therapeutically effective amount of the active ingredientwhen administered as a monotherapy.

The term “treatment” or “treating” means any treatment of a disease ordisorder, including preventing or protecting against the disease ordisorder (that is, causing the clinical symptoms not to develop);inhibiting the disease or disorder (i.e., arresting or suppressing thedevelopment of clinical symptoms; and/or relieving the disease ordisorder (i.e., causing the regression of clinical symptoms). As will beappreciated, it is not always possible to distinguish between“preventing” and “suppressing” a disease or disorder since the ultimateinductive event or events may be unknown or latent. Accordingly, theterm “prophylaxis” will be understood to constitute a type of“treatment” that encompasses both “preventing” and “suppressing”. Theterm “protection” thus includes “prophylaxis”.

The present invention also includes derivatives of the compounds of theinvention, including structural analogues, compounds derived fromcompounds of the invention, prodrug, polymorph forms, metabolites,heat-transformed chemical products, and pharmaceutically acceptablesalts, and combinations thereof.

The present invention also includes other forms of the compounds of theinvention, including prodrug and polymorph forms. Here, a “prodrug” is acompound that contains one or more functional groups that can be removedor modified in vivo to result in a molecule that can exhibit therapeuticutility in vivo. A “polymorph” refers to a compound that has anidentical chemical composition (i.e., it is of the same compoundspecies) as compared to another compound but that differs in crystalstructure. In preferred embodiments, the agents, could be removed ormodified in vivo or comprise the same composition as other natural orsynthetic compounds but differing in specific structure.

Methods of the Invention

The invention provides methods of treating auditory impairments in asubject, e.g., a subject suffering from or experiencing an auditoryimpairment, cochlear inflammation, permanent sensorineural hearing loss,tinnitus, loudness recruitment, hyperacusis, diplacusis or speechintelligibility deficits. In one embodiment, the method comprisesadministering to said subject an effective amount of any of thecompositions of the invention (e.g., a composition comprising as anactive agent one or more of a carboxy alkyl ester, a quinic acidderivative, a caffeic acid derivative, a ferulic acid derivative, or aquinic acid lactone or derivative thereof or pharmaceutically acceptablesalt thereof and an acceptable carrier or excipient) so as to treat anauditory impairment in the subject. In another embodiment, the methodcomprises administering to the subject one or more of a purified orisolated carboxy alkyl ester, purified or isolated quinic acid orderivative, purified or isolated caffeic acid or derivative, a purifiedor isolated ferulic acid or derivative, or a purified or isolated quinicacid lactone or derivative thereof or a pharmaceutically acceptable saltthereof in a sufficient amount so as to treat the auditory impairment.The active agents of the invention may be included in one or morenutraceutical compositions, such as nutritional and/or dietarysupplements.

In a further embodiment of the invention, the method comprisesadministering to said subject an effective amount of a compositioncomprising, as an active agent, one or more of a carboxy alkyl ester,alkaloid, pentacyclic alkaloid, tannin, or phytochemical derived fromthe inner bark or root of Uncaria tomentosa or derivative thereof orpharmaceutically acceptable salt thereof and an acceptable carrier orexcipient so as to treat auditory impairments in the subject. In yetanother embodiment, the active agent is a purified or isolated carboxyalkyl ester, alkaloid, pentacyclic alkaloid, tannin, or phytochemicalderived from the inner bark, leaves or root of Uncaria tomentosa andother plants. For example, the active agents may be found in extracts orextracted from Uncaria tomentosa and used as nutraceuticals or aspharmaceuticals. Nutraceutical includes a composition that may havehealthful effects in a subject upon administration, wherein thecomposition is, for example, available to a subject without a doctor'sprescription.

In one embodiment, the carboxy alkyl ester may be any of (1)3,4-O-dicaffeoylquinic acid; (2) 3,5-O-dicaffeoylquinic acid (CASRegistry Number: 2450-53-5); (3) 1,3-O-dicaffeoylquinic acid (CASRegistry Number: 19870-46-3); (4) 4,5-O-dicaffeoylquinic acid (CASRegistry Number: 32451-88-0); (5) 1,5-O-dicaffeoylquinic acid (CASRegistry Number: 19870-46-3); (6) 3-O-feruloylquinic acid; (7)4-O-feruloylquinic acid; (8) 5-O-feruloylquinic acid; (9)1-O-caffeoylquinic acid; (10) 3-O-caffeoylquinic acid; (11)4-O-caffeoylquinic acid; (12) 5-O-caffeoylquinic acid; (13)(1S,3R,4R,5R)-3-[3-(3,4-dihydroxyphenyl)-3R-hydroxypropanoyl]1,4,5-trihydroxycyclohexanecarboxylicacid; (14) (1S,3R,4R,5R)-3-[3-(3,4dihydroxyphenyl)-3S-hydroxypropanoyl]-1,4,5-trihydroxycyclohexanecarboxylicacid; (15)(1S,3R,4R,5R)-5-[3-(3,4-dihydroxyphenyl)-3R-hydroxypropanoyl]-1,3,4-trihydroxycyclohexanecarboxylicacid; (16)(1S,3R,4R,5R)-5-[3-(3,4-dihydroxyphenyl)-3S-hydroxypropanoyl]-1,3,4-trihydroxycyclohexanecarboxylicacid; (17)(1S,3R,4R,5R)-4-[3-(3,4-dihydroxyphenyl)-3R-hydroxypropanoyl]-1,3,5-trihydroxycyclohexanecarboxylicacid; (18)(1S,3R,4R,5R)-4-[3-(3,4-dihydroxyphenyl)-3S-hydroxypropanoyl]-1,3,5-trihydroxycyclohexanecarboxylicacid; (19) cis-5-O-caffeoylquinic acid; (20) 3-O-caffeoylquinic acidlactone; (21) 3-O-caffeoyl-4-O-feruloylquinic acid; and (22) otherquinic acid esters, and pharmaceutically acceptable salt thereof. Thecarboxy alkyl esters may be included in one or more nutraceuticalcompositions, such as nutritional and/or dietary supplements.

Merely by way of example, the active agents may also be found in anutritional supplement formerly referred to as C-Med-100 and nowcommonly referred to as AC-11 (sold, for example, by Activar Company,Onnit Labs, Solgar Company, Sotrue Company, Optigenex Company andCeregenex Company).

In one embodiment, the composition of the invention further comprisesany one or more of an alkaloid, pentacyclic alkaloid, tannin, or aphytochemical of Uncaria tomentosa or derivative thereof orpharmaceutically acceptable salt thereof.

In another embodiment, the composition may be an aqueous extract fromsaid bark, other plants or plant parts or synthetic derivatives. Someextracts according to the invention may also be provided to a subject asnutraceuticals, for example, as adjuvants in the treatment of a varietyof condition including, but not limited to, auditory dysfunctions (e.g.,hearing loss, tinnitus, hyperacusis, and related auditory processingdisorders).

In one embodiment, the composition may be formulated for administrationselected from the group consisting of auricular, oral, parenteral,intraperitoneal, local, buccal, nasal, and topical administration.

In another embodiment, the composition may be in the form of a liquid,tablet or capsule.

In another embodiment, the hearing impairment may be hearing loss ordeafness. For example, a subject is hearing impaired when the subject'saudiogram shows hearing thresholds greater than 25 dB HL at anyfrequency or the subject exhibits difficulty understanding speech withor without the presence of background noise.

In another embodiment, administration of the composition may be effectedduring or after an insult that can damage the auditory system.

Examples of auditory impairments may include but not limited topermanent sensorineural hearing loss, tinnitus, loudness recruitment,hyperacusis, diplacusis and speech intelligibility deficits.

The invention also provides methods for inhibiting cochlear inflammationin a subject suffering cochlear inflammation. In one embodiment, themethod comprises administering to said subject an effective amount ofany of the compositions of the invention (e.g., a composition comprisingas an active agent one or more of a carboxy alkyl ester, a quinic acidderivative, a caffeic acid derivative, a ferulic acid derivative, or aquinic acid lactone or derivative thereof or pharmaceutically acceptablesalt thereof and an acceptable carrier or excipient) so as to inhibitcochlear inflammation in the subject. In another embodiment, the methodcomprises administering to the subject one or more of a purified orisolated carboxy alkyl ester, purified or isolated quinic acid orderivative, purified or isolated caffeic acid or derivative, a ferulicacid or derivative, or a quinic acid lactone or derivative thereof or apharmaceutically acceptable salt thereof in a sufficient amount so as toinhibit cochlear inflammation.

In a further embodiment of the invention, the method comprisesadministering to said subject an effective amount of a compositioncomprising as an active agent one or more of a carboxy alkyl ester,alkaloid, pentacyclic alkaloid, tannin, or phytochemical derived fromthe inner bark or root of Uncaria tomentosa or derivative thereof orpharmaceutically acceptable salt thereof and an acceptable carrier orexcipient so as to inhibit cochlear inflammation in the subject. In yetanother embodiment, the method comprises administering to the subjectone or more of a purified or isolated carboxy alkyl ester, alkaloid,pentacyclic alkaloid, tannin, or phytochemical derived from the innerbark or root of Uncaria tomentosa or derivative thereof orpharmaceutically acceptable salt thereof in a sufficient amount so as toinhibit cochlear inflammation.

In one embodiment, the method of the invention inhibits hearing loss ina subject by inhibiting cochlear inflammation.

In one embodiment, the composition further comprises any one or more ofan alkaloid, pentacyclic alkaloid, tannin, or a phytochemical of Uncariatomentosa or derivative thereof or pharmaceutically acceptable saltthereof.

The invention also provides a method of inhibiting the loss or death ofthe cells of the auditory system in a subject, e.g., a subject sufferingfrom or experiencing an auditory impairment, cochlear inflammation,permanent sensorineural hearing loss, tinnitus, loudness recruitment,hyperacusis, diplacusis or speech intelligibility deficits.

In one embodiment, the method comprises administering to said subject aneffective amount of any of the compositions of the invention (e.g., acomposition comprising as an active agent one or more of a carboxy alkylester, a quinic acid derivative, a caffeic acid derivative, a ferulicacid derivative, or a quinic acid lactone or derivative thereof orpharmaceutically acceptable salt thereof and an acceptable carrier orexcipient) so as to inhibit the loss or death of the cells of theauditory system in the subject. In another embodiment, the methodcomprises administering to the subject one or more of a purified orisolated carboxy alkyl ester, purified or isolated quinic acid orderivative, purified or isolated caffeic acid or derivative, a ferulicacid or derivative, or a quinic acid lactone or derivative thereof or apharmaceutically acceptable salt thereof in a sufficient amount so as toinhibit the loss or death of the cells of the auditory system.

In a further embodiment of the invention, the method comprisesadministering to said subject an effective amount of a compositioncomprising as an active agent one or more of a carboxy alkyl ester,alkaloid, pentacyclic alkaloid, tannin, or phytochemical derived fromthe inner bark or root of Uncaria tomentosa or derivative thereof orpharmaceutically acceptable salt thereof and an acceptable carrier orexcipient so as to inhibit the loss or death of the cells of theauditory system in the subject. In yet another embodiment, the methodcomprises administering to the subject one or more of a purified orisolated carboxy alkyl ester, alkaloid, pentacyclic alkaloid, tannin, orphytochemical derived from the inner bark or root of Uncaria tomentosaor derivative thereof or pharmaceutically acceptable salt thereof in asufficient amount so as to inhibit the loss or death of the cells of theauditory system.

In one embodiment, the method of the invention inhibits hearing loss ina subject by inhibiting the loss or death of the cells of the auditorysystem.

In another embodiment, administration of the composition may be effectedprior to an insult that can damage the auditory system.

In another embodiment, the composition may be administeredprophylactically over a period of days, weeks, or months.

The invention also provides a method of maintaining or promoting thegrowth of cells of the auditory system of a subject.

In one embodiment, the method comprises administering to said subject aneffective amount of any of the compositions of the invention (e.g., acomposition comprising as an active agent one or more of a carboxy alkylester, a quinic acid derivative, a caffeic acid derivative, a ferulicacid derivative, or a quinic acid lactone or derivative thereof orpharmaceutically acceptable salt thereof and an acceptable carrier orexcipient) so as to maintain or promote the growth of cells of theauditory system in the subject. In another embodiment, the methodcomprises administering to the subject one or more of a purified orisolated carboxy alkyl ester, purified or isolated quinic acid orderivative, purified or isolated caffeic acid or derivative, a ferulicacid or derivative, or a quinic acid lactone or derivative thereof or apharmaceutically acceptable salt thereof in a sufficient amount so as tomaintain or promote the growth of cells of the auditory system.

In a further embodiment of the invention, the method comprisesadministering to said subject an effective amount of a compositioncomprising as an active agent one or more of a carboxy alkyl ester,alkaloid, pentacyclic alkaloid, tannin, or phytochemical derived fromthe inner bark or root of Uncaria tomentosa or derivative thereof orpharmaceutically acceptable salt thereof and an acceptable carrier orexcipient so as to maintain or promote the growth of cells of theauditory system in the subject. In yet another embodiment, the methodcomprises administering to the subject one or more of a purified orisolated carboxy alkyl ester, alkaloid, pentacyclic alkaloid, tannin, orphytochemical derived from the inner bark or root of Uncaria tomentosaor derivative thereof or pharmaceutically acceptable salt thereof in asufficient amount so as to maintain or promote the growth of cells ofthe auditory system.

In one embodiment, the method of the invention inhibits hearing loss ina subject by maintaining or promoting the growth of cells of theauditory system of the subject.

The invention also provides a method of inhibiting or reversing iondyshomeostasis, mitochondriopathy, energy catastrophe and/or theproliferation of free radicals in the auditory system of a subject.

In one embodiment, the method comprises administering to said subject aneffective amount of any of the compositions of the invention (e.g., acomposition comprising as an active agent one or more of a carboxy alkylester, a quinic acid derivative, a caffeic acid derivative, a ferulicacid derivative, or a quinic acid lactone or derivative thereof orpharmaceutically acceptable salt thereof and an acceptable carrier orexcipient) so as to inhibit or reverse ion dyshomeostasis,mitochondriopathy, energy catastrophe and/or the proliferation of freeradicals in the auditory system in the subject. In another embodiment,the method comprises administering to the subject one or more of apurified or isolated carboxy alkyl ester, purified or isolated quinicacid or derivative, purified or isolated caffeic acid or derivative, aferulic acid or derivative, or a quinic acid lactone or derivativethereof or a pharmaceutically acceptable salt thereof in a sufficientamount so as to inhibit or reverse ion dyshomeostasis,mitochondriopathy, energy catastrophe and/or the proliferation of freeradicals in the auditory system.

In a further embodiment of the invention, the method comprisesadministering to said subject an effective amount of a compositioncomprising as an active agent one or more of a carboxy alkyl ester,alkaloid, pentacyclic alkaloid, tannin, or phytochemical derived fromthe inner bark or root of Uncaria tomentosa or derivative thereof orpharmaceutically acceptable salt thereof and an acceptable carrier orexcipient so as to inhibit or reverse ion dyshomeostasis,mitochondriopathy, energy catastrophe and/or the proliferation of freeradicals in the auditory system in the subject. In yet anotherembodiment, the method comprises administering to the subject one ormore of a purified or isolated carboxy alkyl ester, alkaloid,pentacyclic alkaloid, tannin, or phytochemical derived from the innerbark or root of Uncaria tomentosa or derivative thereof orpharmaceutically acceptable salt thereof in a sufficient amount so as toinhibit or reverse ion dyshomeostasis, mitochondriopathy, energycatastrophe and/or the proliferation of free radicals in the auditorysystem.

Compositions of the Invention

This invention also concerns treatment compositions for use in treatingor inhibiting auditory dysfunctions (e.g., hearing loss, tinnitus,hyperacusis, and related auditory processing disorders), particularlynutraceutical, pharmaceutical or veterinary compositions, comprising theagent formulated together with one or more non-toxic acceptablecarriers, preferably pharmaceutically acceptable carriers. The terms“pharmaceutically acceptable carrier” and “physiologically acceptablecarrier” refer to molecular entities and compositions that arephysiologically tolerable and do not typically produce an unintendedallergic or similar untoward reaction, such as gastric upset, dizzinessand the like, when administered to a subject. In the context oftherapeutic compositions intended for human administration,pharmaceutically acceptable carriers are used. The agents may beprocessed in accordance with conventional methods of pharmaceuticalcompounding techniques to produce medicinal agents (i.e., medicaments ortherapeutic compositions) for administration to subjects, includinghumans and other mammals, i.e., “pharmaceutical” and “veterinary”administration, respectively. See, for example, the latest edition ofRemington's Pharmaceutical Sciences (Mack Publishing Co., Easton, Pa.).Typically, a composition such as one or more of the agents are combinedas a composition with a pharmaceutically acceptable carrier. Thecomposition(s) may also include one or more of the following:excipients; preserving agents; solubilizing agents; stabilizing agents;wetting agents; emulsifiers; sweeteners; colorants; odorants; salts;buffers; coating agents; and antioxidants.

The present invention provides pharmaceutical formulations for use intreating or inhibiting auditory dysfunctions (e.g., hearing loss,tinnitus, hyperacusis, and related auditory processing disorders), (alsoknown as pharmaceutical compositions or dosage forms) comprising as itsonly active agent, one or more of a carboxy alkyl ester, alkaloid,pentacyclic alkaloid, tannin, or phytochemical derived from the innerbark or root of Uncaria tomentosa or derivative thereof orpharmaceutically acceptable salt thereof, and a pharmaceuticallyacceptable carrier or vehicle. In another embodiment, the active agentmay be one or more of a carboxy alkyl ester, quinic acid or derivative,caffeic acid or derivative, a ferulic acid or derivative, or a quinicacid lactone or derivative thereof or a pharmaceutically acceptable saltthereof. In an embodiment of the invention, no other active agentsexcept any one or more of those listed supra (or its specificembodiments listed infra) are included in the pharmaceuticalformulation.

Further, the present invention also provides pharmaceutical formulationsfor use in treating or inhibiting auditory dysfunctions (e.g., hearingloss, tinnitus, hyperacusis, and related auditory processing disorders),(also known as pharmaceutical compositions or dosage forms) consistingof as the active agent(s), one or more of a carboxy alkyl ester,alkaloid, pentacyclic alkaloid, tannin, or phytochemical derived fromthe inner bark or root of Uncaria tomentosa or derivative thereof orpharmaceutically acceptable salt thereof, and pharmaceuticallyacceptable carrier(s) or vehicle(s). In another embodiment, thepharmaceutical formulation consists of an active agent(s), which is oneor more of a carboxy alkyl ester, quinic acid or derivative, caffeicacid or derivative, a ferulic acid or derivative, or a quinic acidlactone or derivative thereof or a pharmaceutically acceptable saltthereof. In an embodiment of the invention, no other active agentsexcept those listed supra (or its specific embodiments listed infra) areincluded in the pharmaceutical formulation.

Suitable examples of carboxy alkyl esters include, but are not limitedto, any of (1) 3,4-O-dicaffeoylquinic acid; (2) 3,5-O-dicaffeoylquinicacid (CAS Registry Number: 2450-53-5); (3) 1,3-O-dicaffeoylquinic acid(CAS Registry Number: 19870-46-3); (4) 4,5-O-dicaffeoylquinic acid (CASRegistry Number: 32451-88-0); (5) 1,5-O-dicaffeoylquinic acid (CASRegistry Number: 19870-46-3); (6) 3-O-feruloylquinic acid; (7)4-O-feruloylquinic acid; (8) 5-O-feruloylquinic acid; (9)1-O-caffeoylquinic acid; (10) 3-O-caffeoylquinic acid; (11)4-O-caffeoylquinic acid; (12) 5-O-caffeoylquinic acid; (13)(1S,3R,4R,5R)-3-[3-(3,4-dihydroxyphenyl)-3R-hydroxypropanoyl]1,4,5-trihydroxycyclohexanecarboxylicacid; (14)(1S,3R,4R,5R)-3-[3-(3,4-dihydroxyphenyl)-3S-hydroxypropanoyl]-1,4,5-trihydroxycyclohexanecarboxylicacid; (15)(1S,3R,4R,5R)-5-[3-(3,4-dihydroxyphenyl)-3R-hydroxypropanoyl]-1,3,4-trihydroxycyclohexanecarboxylicacid; (16)(1S,3R,4R,5R)-5-[3-(3,4-dihydroxyphenyl)-3S-hydroxypropanoyl]-1,3,4-trihydroxycyclohexanecarboxylicacid; (17)(1S,3R,4R,5R)-4-[3-(3,4-dihydroxyphenyl)-3R-hydroxypropanoyl]-1,3,5-trihydroxycyclohexanecarboxylicacid; (18)(1S,3R,4R,5R)-4-[3-(3,4-dihydroxyphenyl)-3S-hydroxypropanoyl]-1,3,5-trihydroxycyclohexanecarboxylicacid; (19) cis-5-O-caffeoylquinic acid; (20) 3-O-caffeoylquinic acidlactone; (21) 3-O-caffeoyl-4-O-feruloylquinic acid; and (22) otherquinic acid esters, and pharmaceutically acceptable salt thereof.

Typically, the pharmaceutically acceptable carriers or vehicles willinclude, but are not limited to, binders, diluents, adjuvants,excipients, preserving agents, fillers, polymers, disintegrating agents,glidants, wetting agents, emulsifying agents, suspending agents,sweetening agents, flavoring agents, perfuming agents, lubricatingagents, acidifying agents, coloring agent, dyes, preservatives anddispensing agents, or compounds of a similar nature depending on thenature of the mode of administration and dosage forms. Such ingredients,including pharmaceutically acceptable carriers and excipients that maybe used to formulate oral dosage forms, are described in the Handbook ofPharmaceutical Excipients, American Pharmaceutical Association (1986),incorporated herein by reference in its entirety.

Pharmaceutically acceptable carriers are generally non-toxic torecipients at the dosages and concentrations employed and are compatiblewith other ingredients of the formulation. Examples of pharmaceuticallyacceptable carriers include water, saline, Ringer's solution, dextrosesolution, ethanol, polyols, vegetable oils, fats, ethyl oleate,liposomes, waxes polymers, including gel forming and non-gel formingpolymers, and suitable mixtures thereof. The carrier may contain minoramounts of additives such as substances that enhance isotonicity andchemical stability. Such materials are non-toxic to recipients at thedosages and concentrations employed, and include buffers such asphosphate, citrate, succinate, acetic acid, and other organic acids ortheir salts; antioxidants such as ascorbic acid; low molecular weight(less than about ten residues) polypeptides, e.g., polyarginine ortripeptides; proteins, such as serum albumin, gelatin, orimmunoglobulin; hydrophilic polymers such as polyvinylpyrrolidone; aminoacids, such as glycine, glutamic acid, aspartic acid, or arginine;monosaccharides, disaccharides, and other carbohydrates includingcellulose or its derivatives, glucose, mannose, or dextrins; chelatingagents such as EDTA; sugar alcohols such as mannitol or sorbitol;counterions such as sodium; and/or nonionic surfactants such aspolysorbates, poloxamers, or PEG. Preferably the carrier is a parenteralcarrier, more preferably a solution that is isotonic with the blood ofthe recipient.

Examples of binders include, but are not limited to, microcrystallinecellulose and cellulose derivatives, gum tragacanth, glucose solution,acacia mucilage, gelatin solution, molasses, polyvinylpyrrolidine,povidone, crospovidones, sucrose and starch paste.

Examples of diluents include, but are not limited to, lactose, sucrose,starch, kaolin, salt, mannitol and dicalcium phosphate.

Examples of excipients include, but are not limited to, starch,surfactants, lipophilic vehicles, hydrophobic vehicles, pregelatinizedstarch, Avicel, lactose, milk sugar, sodium citrate, calcium carbonate,dicalcium phosphate, and lake blend purple. Typical excipients fordosage forms such as a softgel include gelatin for the capsule and oilssuch as soy oil, rice bran oil, canola oil, olive oil, corn oil, andother similar oils; glycerol, polyethylene glycol liquids, vitamin ETPGS as a surfactant and absorption enhancer (Softgels: ManufacturingConsiderations; Wilkinson P, Foo Sog Hom, Special Drug Delivery Systems;Drugs and the Pharmaceutical Sciences Vol 41 Praveen Tyle Editor, MarcelDekker 1990, 409-449; Pharmaceutical Dosage Forms and Drug Delivery byAnsel, Popovich and Allen 1995, Williams and Wilkins, Chapter 5 pp155-225). Tritoqualine and anti H1 may form either a solution in aselected oil vehicle or a suspension of fine particles (comprising anyof the excipients disclosed herein, e.g., typical excipients forsoftgels).

Examples of disintegrating agents include, but are not limited to,complex silicates, croscarmellose sodium, sodium starch glycolate,alginic acid, corn starch, potato starch, bentonite, methylcellulose,agar and carboxymethylcellulose.

Examples of glidants include, but are not limited to, colloidal silicondioxide, talc, corn starch.

Examples of wetting agents include, but are not limited to, propyleneglycol monostearate, sorbitan monooleate, diethylene glycol monolaurateand polyoxyethylene laural ether.

Examples of sweetening agents include, but are not limited to, sucrose,lactose, mannitol and artificial sweetening agents such as saccharin,and any number of spray dried flavors.

Examples of flavoring agents include, but are not limited to, naturalflavors extracted from plants such as fruits and synthetic blends ofcompounds which produce a pleasant sensation, such as, but not limitedto peppermint and methyl salicylate.

Examples of lubricants include magnesium or calcium stearate, sodiumlauryl sulphate, talc, starch, lycopodium and stearic acid as well ashigh molecular weight polyethylene glycols.

Examples of coloring agents include, but are not limited to, any of theapproved certified water soluble FD and C dyes, mixtures thereof; andwater insoluble FD and C dyes suspended on alumina hydrate.

The artisan of ordinary skill in the art will recognize that manydifferent ingredients can be used in formulations according to thepresent invention, in addition to the active agents, while maintainingeffectiveness of the formulations in treating the auditory dysfunctions(e.g., hearing loss, tinnitus, hyperacusis, and related auditoryprocessing disorders). The list provided herein is not exhaustive.

Further still, the agents, and their respective acid or base salts, canbe formulated into liquid, preferably aqueous, formulations for storageand administration, as well as dried formulations that may, for example,be used as powders for intranasal or oral administration or bereconstituted into liquid form just prior to administration to asubject. Liquid pharmaceutically administrable compositions can, forexample, be prepared by dissolving, dispersing, etc. the particularagent and optional pharmaceutical adjuvants in an aqueous carrier.Aqueous carriers include water (particularly water for injection intohumans), alcoholic/aqueous solutions, and emulsions and suspensions.Preferred pharmaceutically acceptable aqueous carriers include sterilebuffered isotonic saline solutions. Parenteral vehicles include sodiumchloride solution, Ringer's dextrose, dextrose, and sodium chloride,lactated Ringer's, or fixed oils. Intravenous vehicles include fluid andnutrient replenishers, electrolyte replenishers (such as those based onRinger's dextrose), and the like. Preservatives and other additives mayalso be present, such as, for example, antimicrobials, antioxidants,chelating agents, and inert gases and the like. Non-aqueous solvents mayalso be included, although when included they preferably comprise lessthan about 50%, more preferably less than about 25%, and even morepreferably less about 10%, of the total solvent of the solution.Examples of non-aqueous solvents include propylene glycol, ethanol,polyethylene glycol, vegetable oils such as olive oil, and injectableorganic esters such as ethyl oleate. The neutraceutical, pharmaceuticaland veterinary compositions of the agents, whether dry or liquid, arepreferably formulated for oral administration.

If desired, the composition to be administered may also contain minoramounts of nontoxic auxiliary carrier or excipient substances such aswetting agents, emulsifying agents, or solubilizing agents,antioxidants, antimicrobials, pH buffering agents and the like, forexample, sodium acetate, sodium citrate, cyclodextrin derivatives,sorbitan monolaurate, triethanolamine acetate, triethanolamine oleate,etc. Actual methods of preparing such dosage forms are known, or will beapparent, to those skilled in this art; for example, see Remington: TheScience and Practice of Pharmacy, Mack Publishing Company, Easton, Pa.,20th Edition, 2000. The composition or formulation to be administeredwill, in any event, contain a quantity of the active compound in anamount effective to alleviate the symptoms of the subject being treated.

As those in the art will appreciate, the agents of the invention mayalso be formulated for targeted delivery to a subset of tissues or cellsin a subject. In general, targeted delivery is accomplished byformulating a compound of the agents with a targeting moiety. Suchmoieties include lipids, liposomes, and ligands for molecules that bind,or are bound by, other molecules in vivo.

Any derived form of the agents (example synthetic or natural), or aconjugate thereof, can be prepared as an acid salt or as a base salt, aswell as in free acid or free base forms. Such compositions if used toprevent or treat auditory dysfunctions are covered under the preferredembodiment of this invention.

The amount of the agent required for use in treatment will vary not onlywith the particular agent and salt selected, but also with the route ofadministration, the nature of the condition being treated, and the ageand condition of the patient, among other factors, and ultimately isdetermined at the discretion of the attending physician or clinician.The desired dose may conveniently be presented in a single dose or asdivided doses administered at appropriate intervals, for example, astwo, three, four or more sub-doses per day. The sub-dose itself may befurther divided, for example, into a number of discrete, loosely spacedadministrations, such as multiple ingestations of pill doses, or liquiddoses.

Administration

The agents of this invention may be administered in a therapeuticallyeffective amount to a subject in need of treatment. Administration ofcompositions of the invention can be via any of suitable route ofadministration, particularly by ingestion, or alternativelyparenterally, for example, intratympanically, intravenously,intra-arterially, intraperitoneally, intrathecally, intraventricularly,intraurethrally, intrasternally, intracranially, intramuscularly,intranasally, subcutaneously, sublingually, transdermally, or byinhalation or insufflations, or topical by ear instillation forabsorption through the skin of the ear canal and membranes of theeardrum. Such administration may be as a single oral dose, definednumber of ear drops, or a bolus injection, multiple injections, or as ashort- or long-duration infusion. Implantable devices (e.g., implantableinfusion pumps) may also be employed for the periodic parenteraldelivery over time of equivalent or varying dosages of the particularformulation. For such parenteral administration, the compounds arepreferably formulated as a sterile solution in water or another suitablesolvent or mixture of solvents. The solution may contain othersubstances such as salts, sugars (particularly glucose or mannitol), tomake the solution isotonic with blood, buffering agents such as acetic,citric, and/or phosphoric acids and their sodium salts, andpreservatives. The preparation of suitable, and preferably sterile,parenteral formulations is described in detail in the section entitled“Compositions”, above.

In the context of this invention, actual dosage levels for thecompositions of the invention can be varied so as to obtain an amount ofthe active agent(s) that is effective to achieve the desired therapeuticresponse for a particular patient, compositions and mode ofadministration. In general, daily administration or continuous infusionat dosages less than those known to produce toxicities will be thepreferred therapeutic protocol to enhance the activity of the agent(s).The selected dosage level will depend upon the activity of theparticular agent(s), the route of administration, the severity of thecondition being treated and the condition and prior medical history ofthe patient being treated. However, it is within the skill of the art tostart doses of the compound at levels lower than required to achieve thedesired therapeutic effect and to gradually increase the dosage untilthe desired effect is achieved.

With regard to human and veterinary treatment, the amount of aparticular agent(s) that is administered will, of course, be dependenton a variety of factors, including the disorder being treated and theseverity of the disorder; activity of the specific agent(s) employed;the age, body weight, general health, sex and diet of the patient; thetime of administration, route of administration, and rate of excretionof the specific agent(s) employed; the duration of the treatment; drugsused in combination or coincidental with the specific agent(s) employed;the judgment of the prescribing physician or veterinarian; and likefactors well known in the medical and veterinary arts.

In further embodiments, the agent(s) comprises treatment formulationsthat can be made in powdered form for administration via ingestion, withor without additional ingredients, such as dietary supplements,comprising combination formulas with vitamins, minerals and othernutritional supplements.

Dosage forms include tablets, troches, dispersions, suspensions,solutions, capsules, patches, and the like. The agent(s) may beadministered prior to, concurrently with, or after administration ofother auditory therapies, or continuously, i.e., in daily doses, duringall or part of, a separate auditory therapy regimen. The agent, in somecases, may be combined with the same carrier or vehicle used to deliverthe other auditory therapy.

Thus, the present agent(s) may be systemically administered, e.g.,orally, in combination with a pharmaceutically acceptable vehicle suchas an inert diluent or an assimilable edible carrier. The agent(s) maybe enclosed in hard or soft shell gelatin capsules, may be compressedinto tablets, or may be incorporated directly with the food of thepatient's diet. For oral therapeutic administration, the agent(s) may becombined with one or more excipients and used in the form of ingestibletablets, buccal tablets, troches, capsules, elixirs, suspensions,syrups, wafers, and the like.

The tablets, troches, pills, capsules, and the like may also contain thefollowing: binders such as gum tragacanth, acacia, corn starch orgelatin; excipients such as dicalcium phosphate; a disintegrating agentsuch as corn starch, potato starch, alginic acid and the like; alubricant such as magnesium stearate; and a sweetening agent such assucrose, fructose, lactose or aspartame or a flavoring agent such aspeppermint, oil of wintergreen, or cherry flavoring may be added. Whenthe unit dosage form is a capsule, it may contain, in addition tomaterials of the above type, a liquid carrier, such as a vegetable oilor a polyethylene glycol. Various other materials may be present ascoatings or to otherwise modify the physical form of the solid unitdosage form. For instance, tablets, pills, or capsules may be coatedwith gelatin, wax, shellac or sugar and the like. Tablets, capsules,pills, granules, microparticles and the like can also comprise anenteric coating, such as a coating of one of the Eudragit® polymers,that will permit release of the agent(s) in the intestines, not in theacidic environment of the stomach.

A syrup or elixir may contain the active compound, sucrose or fructoseas a sweetening agent, methyl and propylparabens as preservatives, a dyeand flavoring such as cherry or orange flavor. Of course, any materialused in preparing any unit dosage form should be pharmaceuticallyacceptable and substantially non-toxic in the amounts employed. Inaddition, the agent(s) may be incorporated into sustained-releasepreparations and devices.

The agent(s) or their compositions may also be administeredintravenously or intraperitoneally by infusion or injection. Solutionsof the agent(s) or its salts can be prepared in water, optionally mixedwith a non-toxic surfactant. Dispersions can also be prepared inglycerol, liquid polyethylene glycols, triacetin, and mixtures thereofand in oils. Under ordinary conditions of storage and use, thesepreparations contain a preservative to prevent the growth ofmicroorganisms.

The pharmaceutical dosage forms suitable for injection or infusion caninclude sterile aqueous solutions or dispersions or sterile powderscomprising the active ingredient which are adapted for theextemporaneous preparation of sterile injectable or infusible solutionsor dispersions, optionally encapsulated in liposomes. In all cases, theultimate dosage form must be sterile, fluid and stable under theconditions of manufacture and storage. The liquid carrier or vehicle canbe a solvent or liquid dispersion medium comprising, for example, water,ethanol, a polyol (for example, glycerol, propylene glycol, liquidpolyethylene glycols, and the like), vegetable oils, non-toxic glycerylesters, and suitable mixtures thereof. The proper fluidity can bemaintained, for example, by the formation of liposomes, by themaintenance of the required particle size in the case of dispersions orby the use of surfactants. The prevention of the action ofmicroorganisms can be brought about by various antibacterial andantifungal agents, for example, parabens, chlorobutanol, phenol, sorbicacid, thimerosal, and the like. In many cases, it will be preferable toinclude isotonic agents, for example, sugars, buffers or sodiumchloride. Prolonged absorption of the injectable compositions can bebrought about by the use in the compositions of agents delayingabsorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions can be prepared by incorporating theagent(s) in the required amount in the appropriate solvent with variousother ingredients enumerated above, as required, followed by filtersterilization. In the case of sterile powders for the preparation ofsterile injectable solutions, the preferred methods of preparation arevacuum drying and the freeze drying techniques, which yield a powder ofthe agent(s) plus any additional desired ingredient present in thepreviously sterile-filtered solutions.

The present agent(s) may be locally administered, e.g., topically.

For topical administration, the agent(s) may be applied in liquid orcream-based formulations, which preferably will include adermatologically acceptable carrier, which may be a solid, gel, orliquid. Useful solid carriers include finely divided solids such astalc, clay, microcrystalline cellulose, silica, alumina and the like.Useful liquid carriers include water, alcohols, or glycols orwater-alcohol/glycol blends, in which the agent(s) can be dissolved ordispersed at effective levels, optionally with the aid of non-toxicsurfactants. Adjuvants such as fragrances and additional antimicrobialagents can be added to optimize the properties for a given use. Theresultant liquid compositions can be applied from absorbent pads, usedto impregnate bandages and other dressings, or sprayed onto the affectedarea using pump-type or aerosol sprayers, or dripped or flowed into theear canal using ear droppers or the like. Thickeners such as syntheticpolymers, fatty acids, fatty acid salts and esters, fatty alcohols,modified celluloses, and/or modified mineral materials can also beemployed with liquid carriers to form spreadable pastes, gels,ointments, soaps, and the like, for application directly to the skin ofthe user.

Useful dosages of the agent(s) of the invention can be determined bycomparing their in vitro activity, and in vivo activity in animalmodels. Methods for the extrapolation of effective dosages in mice, andother animals, to humans are known to the art. See, e.g., U.S. Pat. No.4,938,949.

Other drugs or treatments, including treatment with other agents such aschemotherapeutic agents, irradiation, or other anti-cancer agents suchas alkylating agents, anti-tumor antibodies, or cytokines, can be usedwith the present agent(s). See, e.g., Remington's PharmaceuticalSciences (18^(th) ed. 1990) at pages 1138-1162.

In further embodiments, the agent(s) further provides a treatment forhearing injury by acting, in part, to stimulate endogenous protectivemechanisms, particularly in cells involved in hearing. The capacity ofthe agent(s) to induce DNA repair is well documented. For example, ShengY, et al., (DNA repair enhancement of aqueous extracts of Uncariatomentosa in a human volunteer study. Phytomedicine. 2001; 8: 275-283)discloses that in a randomized age and gender matched human study, twogroups of subjects were treated with either 250 or 350 mg tabletscomprising agent(s) extracted from Uncaria tomentosa bark for 8consecutive weeks and compared against a control group not receiving theextract composition. Blood samples comprising blood cells were takenfrom each group and exposed to hydrogen peroxide to induce free radicalDNA damage. Results proved a statistically significant increase inenzymatic DNA repair activity as evidenced by higher levels of repairedDNA single strand breaks in the groups that received the extractedagent(s) as compared to the control group. A similar study was conductedin an animal model (Sheng Y, et al., Enhanced DNA repair, Immunefunction and reduced toxicity of C-MED-100, a novel aqueous extract fromUncaria tomentosa. J Ethnopharmacol. 2000; 69: 115-126) wherein femaleW/Fu rats were treated with the extracted agent(s) in doses up to 160mg/kg body weight, for four weeks by gastric gavage then exposed to 12Gy of whole body irradiation to induce wide-spread genomic damage. Theresults proved significantly (p<0.05) improved DNA repair capacityrelative to control rats not treated with the extract composition. Inembodiments of the invention related to treating auditory dysfunctions,whether treating specifically hearing loss, or alternatively preventionof hearing loss such as in the case of treating a person subject toconstant noise or high noise levels, or such as in the case of a cancerpatient subject to exposure of ototoxic drugs, the current inventioncontemplates administration of the active agent(s) to a patient forproviding protection to the auditory system.

Regarding isolation and preparation of the agent(s). Agent(s) of theinvention may be produced by chemical synthesis or biochemicalsynthesis. Agent(s) of the invention may be obtained by extraction orextraction followed by purification from plants, e.g., Uncariatomentosa, microbes, or genetically engineered organisms. Synthesized,extracted, or purified agent(s) may be subject to further chemicalmodifications or salt formation so long as the intended protective ortherapeutic benefit to the auditory system exists in the modifiedagent(s) or salt. Agent(s) or modified agent(s) of the invention may beused to prepare formulations suitable for the particular route ofadministration, as discussed above. The extracts of the presentinvention may be included in one or more nutraceutical compositions,such as nutritional and/or dietary supplements and food additives, orone or more pharmaceutical compositions.

Applications

As described above, certain aspects of the invention relate tocompositions that contain an agent(s) of the invention, which are usefulin the treatment or prevention of hearing loss or other auditorydysfunctions in, humans, non-human primates (e.g., monkeys, chimpanzees,gorillas and lemurs) or other mammals (e.g., bovine, canine, equine,feline, ovine, murine and porcine animals), and perhaps other animals aswell.

In the context of hearing therapy, the agent(s) of the present inventionmay be used alone, i.e., in monotherapy, or in combination with othertherapeutic agents such as, for example, anti-cancer therapies (e.g.,radiation, surgery, bone marrow transplantation, etc.), that involve useof drugs that are potentially detrimental to hearing or cells associatedwith hearing. As will be appreciated, “combination therapy” (in thecontext of hearing and other therapies) and the like refer to a courseof therapy that involves the provision of at least two distincttherapies to achieve an indicated therapeutic effect. For example, acombination therapy may involve the administration of two or morechemically distinct active ingredients, for example, a fast-actingchemotherapeutic drug and the agent(s). The drugs may be delivered ormay be administered as part of the same composition or as differentcompositions according to the same therapeutic regimen or differentregimens, depending on the active ingredients involved, the disease tobe treated, the age and condition of the patient, etc. Moreover, whenused in combination with another therapeutic agent, the administrationof the two agents may be simultaneous or sequential. Simultaneousadministration includes the administration of a single dosage form thatcomprises both agents, and the administration of the two agents inseparate dosage forms at substantially the same time. Sequentialadministration includes the prior, concurrent, or subsequentadministration of the two or more agents according to the same ordifferent schedules, provided that there is an overlap in the periodsduring which the treatment is provided. Alternatively, a combinationtherapy may involve the administration of one or more of the agents ofthe invention as well as the delivery of radiation therapy and/orsurgery or other techniques to either improve the quality of life of thepatient.

Kits

In a further embodiment, the present invention provides kits (i.e., apackaged combination of reagents with instructions) containing theactive agents of the invention useful for treating an auditoryimpairment, cochlear inflammation, permanent sensorineural hearing loss,tinnitus, loudness recruitment, hyperacusis, diplacusis or speechintelligibility deficits.

The kit can contain a pharmaceutical composition that includes one ormore agents of the invention effective for treating an auditoryimpairment, cochlear inflammation, permanent sensorineural hearing loss,tinnitus, loudness recruitment, hyperacusis, diplacusis or speechintelligibility deficits and an acceptable carrier or adjuvant, e.g.,pharmaceutically acceptable buffer, such as phosphate-buffered saline,Ringer's solution or dextrose solution. It may further include othermaterials desirable from a commercial and user standpoint, includingother buffers, diluents, filters, needles, syringes, and package insertswith instructions for use.

The agents may be provided as dry powders, usually lyophilized,including excipients that upon dissolving will provide a reagentsolution having the appropriate concentration.

The kit comprises one or more containers with a label and/orinstructions. The label can provide directions for carrying out thepreparation of the agents for example, dissolving of the dry powders,and/or treatment for an auditory impairment, cochlear inflammation,permanent sensorineural hearing loss, tinnitus, loudness recruitment,hyperacusis, diplacusis or speech intelligibility deficits.

The label and/or the instructions can indicate directions for in vivouse of the pharmaceutical composition. The label and/or the instructionscan indicate that the pharmaceutical composition is used alone, or incombination with another agent to treat an auditory impairment, cochlearinflammation, permanent sensorineural hearing loss, tinnitus, loudnessrecruitment, hyperacusis, diplacusis or speech intelligibility deficits.

The label can indicate appropriate dosages for the agents of theinvention as described supra.

Suitable containers include, for example, bottles, vials, and testtubes. The containers can be formed from a variety of materials such asglass or plastic. The container can have a sterile access port (forexample the container can be an intravenous solution bag or a vialhaving a stopper pierceable by a needle such as a hypodermic injectionneedle).

In one embodiment, the active agent, alkaloid, pentacyclic alkaloid,tannin or phytochemical may be less than 10,000 molecular weight.

In another embodiment, the active agent, alkaloid, pentacyclic alkaloid,tannin or phytochemical may be hydrophilic.

In yet another embodiment, the active agent, alkaloid, pentacyclicalkaloid, tannin or phytochemical or the aqueous extract may be spraydried on maltodextrin.

The following examples are presented to illustrate the present inventionand to assist one of ordinary skill in making and using the same. Theexamples are not intended in any way to otherwise limit the scope of theinvention.

EXAMPLES Example 1

Examples of the protective effect of a formulation of the agents areshown in FIGS. 1 to 3. FIG. 1 shows distortion product otoacousticemission (DPOAE) levels as a function of frequency for each treatmentgroup; (A) control (water gavage-only), (B) noise exposure-only (nogavage), (C) CAE-only and (D) CAE+noise. Note: CAE (carboxy alkylesters) is a formulation of the agent. Within each treatment group DPOAElevels were measured at four time points; baseline (before anyexperimental treatment) and 1-day, 1-week and 4-weeks post-noiseexposure. The control group and the CAE-only group did not receive noiseexposure. The gray bar represents the frequency range of the damagingnoise (8-kHz OBN at 105 dB SPL for 4 hours). Note that the noise-onlygroup showed depressed DPOAE levels out to 4-weeks after noise exposurewhile the CAE+noise group showed almost complete recovery as early as 1week following the noise exposure. Errors bars are standard errors ofthe means. In this and FIGS. 2 to 4, the data were taken from thepublication, Guthrie et al., Brain Research, 1407; 97-106: 2011.

FIG. 2A shows a photomicrograph of the peripheral auditory neurosensoryepithelium. Outer hair cell (OHC) counts (called cytocochleograms) aredisplayed for (B) control (water gavage-only), (C) noise exposure-only(no gavage), (D) CAE-only (no noise exposure) and (E) CAE+noise treatedgroups. These cytocochleograms reveal the percent of missing OHCs (seearrows in the photomicrograph) from rows 1-3 as a function of distance(0-9 mm) and frequency (1-50 kHz) along the basilar membrane. The noiseexposure was an 8-kHz OBN at 105 dB SPL for 4 hours. Note that the noiseexposure produced OHCs loss, however the level of missing OHCs is lessin the CAE+noise group compared to the noise-only group. Errors bars arestandard errors of the means.

FIG. 3 shows compound action potential (CAP) recordings of neuralsensitivity in dB SPL for control (water gavage-only), noiseexposure-only (no gavage), CAE-only (no noise exposure) and CAE+noisetreated groups. The gray bar represents the frequency range of thedamaging noise (8-kHz OBN at 105 dB SPL for 4 hours). Note that theCAE+noise group exhibit better neural sensitivity than the noise-onlygroup. All CAP recordings were acquired 4-weeks following noise exposureto assess permanent damage. The frequencies tested ranged from 2.0-40.0kHz. Errors bars are standard errors of the means.

Uncaria tomentosa (UT) also known as “uña de gato” or cat's claw is amultifunctional medicinal vine that has been used for over 2000 years byancient civilizations including that of the Tahuantinsuyo (Inca) empire(Pilarski et al., 2005). The bioactive components of UT can be dividedinto hydrophobic and hydrophilic chemotypes (Desmarchelier et al., 1997;Pilarski et al., 2005). The hydrophobic chemotypes include uncarine F,speciophylline, mitraphylline, isomitraphylline, pteropodine andisopteropodine (Bacher et al., 2006; Laus, 2004; Pilarski et al., 2005;Wagner et al., 1985). These hydrophobic chemotypes are derived fromtincture preparations and have received considerable attention for theirrole in immunomodulation, antimicrobial defense, anti-inflammation andantimutagenicity (Keplinger et al., 1999). However, these hydrophobicchemotypes are not representative of medicinal decoctions consumed byancient and indigenous peoples. For instance, the Asháninka Indians ofthe Amazon basin typically boiled UT in water and consumed the resultinghydrophilic chemotypes (Keplinger et al., 1999; Mammone et al., 2006).Recent experiments have demonstrated that carboxy alkyl esters (CAEs;see FIG. 4) are the bioactive components of these hydrophilic chemotypes(Akesson et al., 2005; Sheng et al., 2005). The documented healthbenefits of CAEs include: antioxidant protection, augmentation of DNArepair, anti-inflammation and immunomodulation (Akesson et al., 2003a,2003b; Sandoval et al., 2002). These and other health benefits are basedon the potency of CAEs to potentiate several biochemical cascades inorder to increase the overall capacity of cells to survive and maintainfunctional integrity (Pero, 2010; Pero et al., 2009; Pero and Lund,2010). Furthermore current human, animal and in vitro research hassupported a role for CAEs in augmenting cellular repair from variousphysical or chemical exposures (Akesson et al., 2003a, 2003b; Belkaid etal., 2006; Gurrola-Díaz et al., 2010; Lemaire et al., 1999; Mammone etal., 2006; Pero et al., 2002). However, a role for CAEs in preservingauditory function following noise injury has not been studied. Exposureto high levels of sound may induce a multiplicative array of biochemicalcascades that perpetuate cell death and/or loss of auditory function (LePrell et al. 2007; Ohlemiller, 2008). These biochemical cascades maypropagate within minutes following exposure and are driven by processessuch as ionic dyshomeostasis, mitochondriopathy, energy catastrophe andthe proliferation of free radicals. For instance, loud-sound exposuremay alter cochlear homeostasis of Ca2+, K+, and Na+ particularly throughglutamate excitotoxicity (Hakuba et al., 2000; Le Prell et al. 2007).Mitochondriopathy is evidenced by sound induced increase inmitochondrial permeability and the independent release of at least twomitochondrial nucleases, endonuclease-G and apoptosis inducing-factor(Han et al., 2006; Yamashita et al., 2004b). Furthermore, it is knownthat loud-sound exposure activates the mitochondria-mediatedcaspase-dependent cell death pathway (Nicotera et al., 2003; Wang etal., 2007). Energy catastrophe relates to depleted stores of high energyphosphates (e.g., ATP) following loud-sound exposure (Minami et al.,2007). The proliferation of free radicals is exemplified by increasedproduction of reactive lipid, oxygen and nitrogen species (Ohlemiller etal., 1999; Yamashita et al., 2004a). These combined processes (ionicdyshomeostasis, mitochondriopathy, energy catastrophe and free radicalproduction) complement each other to elicit acute and chronicinflammation that ultimately results in cell death and/or loss ofauditory function (Masuda et al., 2006; Ohlemiller, 2008). The clinicalmanifestation of this combinatorial process includes permanentsensorineural hearing loss, tinnitus, loudness recruitment, hyperacusis,diplacusis and speech intelligibility deficits (Basta et al., 2005;Pienkowski and Eggermont, 2010). These auditory impairments reduce anindividual's quality of life and work productivity such that theeconomic burden to society may average $297,000 over an individual'slife span (Mohr et al., 2000). A major goal in audiologic rehabilitationand neurootologic medicine is the development of biomedical strategiesthat preserve auditory sensory and/or neural function followingloud-sound exposure. To this end an impressive mosaic of pharmaceuticalsthat target individual pathophysiologic cascades has been employed (LePrell et al. 2007; Ohlemiller, 2008). For instance, Ca2+ blockers havebeen used to regulate ionic homeostasis, creatine supplementation hasbeen employed to restore energy and several types of free radicalscavengers have been tested (Minami et al., 2007; Shen et al., 2007).Unfortunately, none of these single-target approaches has gainedwide-spread clinical acceptance due in part to inconsistent outcomes.Since loud-sound exposure induces multiple pathologic cascades, then, analternative approach might be to employ a multifunctional agent thatsimultaneously targets several pathophysiologic mechanisms. Given thatCAEs of UT have demonstrated efficacy as a multifunctionalcytoprotective agent in both human and animal studies, we speculatedthat CAEs might be otoprotective. Therefore, in the current experimentwe tested the hypothesis that CAEs of UT will augment recovery ofsensory and neuronal functions following noise injury.

Materials

CAEs of UT were prepared by Optigenex Inc. (Hoboken, N.J., USA) asAC-110 (also known as C-MED-100®). The extraction and purificationprotocol followed a patented (U.S. Pat. Nos. 6,039,649; 6,238,675 B1;6,361,805 B2 and 6,964,784 B2) process that partly mimics the extractionprocedure used by the Asháninka Indians. The procedure has beenpublished previously (Sheng et al., 2000a, 2000b, 2005). Briefly, thebark (˜150 g) of UT is heated in water for 12-24 h at 90-100° C. and thesoluble extracts are decanted and ultra-filtered to remove componentswith a molecular weight that is greater than 10 kDa (e.g., tannins andflavonoids) while the remaining low molecular weight components arespray dried on maltodextrin. Spray dried maltodextrin may be used tomanufacture dosage forms for the desired routes of administration, e.g.,oral, inhalation, parenteral injection, or topical applications. Bartoschemistry was used to demonstrate the presence of CAEs (Bartos, 1980).For instance, the extracts react with hydroxylamine (10% hydroxylaminehydrochloride in methanol, 10% sodium hydroxide in methanol, pH 10) toproduce hydroxamic acid which was then reacted with ferric chloride(0.3% ferric chloride hexahydrate) to exhibit a chromophore withabsorbance at 490 nm (Lamm et al., 2001; Sheng et al., 2005). To furtherconfirm the presence of CAE, the extracts produced a 200 nm UVabsorption maxima which was standardized against dioctyl phthalate, atypical benzoic acid-type CAE (Sheng et al., 2005). Lastly, a NaOHneutralization procedure served as a third method to verify the presenceof CAE. Here NaOH is used to neutralize the extracts in order todetermine the base equivalents needed to adjust the pH to 7. Up to 20%of the extracts are CAEs which are the only bioactive constituents(Mammone et al., 2006; Sheng et al., 2005). In the current experiment,the extracts (Optigenex, lot #280809.1785) contained 10.25% CAE. Allanimals in the CAE treatment groups were treated with this particularlot of extracts.

Animals

Thirty pigmented male Long-Evans rats (250-300 g at 2 months old) wereacquired from Harlan Laboratories, Inc. (Livermore, Calif., USA) andserved as subjects in these experiments. The animals were housed at theVeterinary Medical Unit (VMU) at the Loma Linda Veteran's Hospital (LomaLinda, Calif., USA). The VMU is accredited by the Association forAssessments and Accreditation of Laboratory Animal Care (AAALAC) and isstaffed with a medical veterinarian and veterinary technicians. Theanimals were maintained in a low-stress and physically-enrichedenvironment where they had free access to food and water. Theenvironmental temperature was maintained at 21° C.±1° C. and thelighting followed a 12 hour light/dark cycle (6:30 am to 6:30 pm). Allexperimental protocols were conducted during the light cycle and eachprotocol was approved by the Hospital's Institutional Animal Care andUse Committee. The experimental protocols were designed to minimize thenumber of animals used, pain and discomfort. Table 1 describes thedifferent animal groups, their treatment regimen and the experimentaldesign. After arriving from Harlan the animals were given 1 week toacclimatize to the VMU. Baseline DPOAEs were then collected on eachanimal to verify auditory function. The animals were then assigned toone of four groups based on their DPOAE measurements to counterbalanceauditory function between groups. CAEs were dissolved indouble-distilled water at a concentration of 160 mg/mL to produce ahomogenous solution suitable for gastric gavage (Sheng et al., 2000a). A20-gage animal feeding stainless steel needle was used to intubate alertanimals in order to administer 160 mg of the CAE solution per kilogramof animal weight. Fresh solutions were prepared each day andadministered via gastric intubation for 28 consecutive days. A controlgroup of animals received water vehicle (volume/body weight) via gastricintubation instead of the CAE solution. Two groups (CAE+noise andnoise-only) were exposed to noise (see Noise exposure) on the 29th day.DPOAE was measured again at 1 day, 1 week and 4 weeks following thenoise exposure. CAP recordings and tissue collection for hair cellcounts (cytocochleograms) were obtained at 4 weeks (end of the study)post-noise exposure.

TABLE 1 Experimental design. 1-day, 1-week and 4 week post-noiseexposure data collection (DPOAE + Baseline CAP + DPOAE 28 days Noisehair cell Groups testing of gavage exposure count) CAE + noise + CAE 105dB OBN + CAE-only + CAE + Noise only + 105 dB OBN + Control + Water +Abbreviations: CAE, carboxy alkyl ester; DPOAE, distortion productotoacoustic emission; CAP, compound action potential; 105 dB OBN, 105decibel (dB) octave band noise centered at 8 kHz for 4 h.

Noise Exposure

In order to elicit noise induced auditory dysfunction, the animals inthe CAE+noise and noise-only groups were exposed to an 8 kHz OBN at 105dB SPL for 4 h. This noise exposure exceeds the permissible doses forwork-place safety in the United States and is known to produce permanentsensorineural auditory dysfunction in rats (Chen and Fechter, 2003;Lorito et al., 2006). Awake and alert animals were placed in a smallwire-cloth enclosure (15×13×11 cm) within a reverberant 40 L chamber.Broadband noise was driven by a DS335 Function Generator (StanfordResearch System, Menlo Park, Calif., USA) and bandpass filtered with aFrequency Device 9002-Dual-Channel Filter/Amplifier Instrument(Frequency Device Inc., Haverhill, Mass., USA) with a roll-off of 48dB/octave to produce an OBN with center frequency at 8 kHz. This OBN wasthen amplified by a HCA1000A Parasound Amplifier (Parasound Products,Inc., San Francisco, Calif., USA) and delivered to Vifa D25AG-05speakers (Vifa International A/S, Videbaek, Denmark) locatedapproximately 5 cm above the animals' wire-cloth enclosure. Soundpressure levels measured at the rats' pinnae were 105 dBlin SPL in theoctave band centered around 8 kHz. These sound pressure measurementswere made using an OB-300Quest Type-1 Sound Pressure Meter with ⅓ octavefilter set (Quest Electronics, Oconomowoc, Wis., USA).

Assessment of Sensory Function

DPOAE was used to assess the function of the OHCs in the right ear. Eachanimal was lightly anesthetized with ketamine (44 mg/kg) and xylazine (7mg/kg) while normal body temperature was maintained using a directcurrent (dc) heating unit built into the surgical table. Allmeasurements were obtained in a double-walled sound-isolation chamber(Industrial Acoustics Company Inc., Bronx, N.Y., USA). The cubic 2f1-f2DPOAE was recorded with two primaries, f2 and f1; where f2 is basal tof1 at an f2/f1 ratio of 1.25. The f1 and f2 frequencies were sweptbasalward in 0.1-octave increments of f2 along the cochlear spiral fromf2=3.2 to 63 kHz. The sound pressure level (SPL) for the f1 primary was65 dB SPL (L1) and that for the f2 primary was 55 dB SPL (L2) with alevel ratio of 1.18 (L1/L2). These combined frequency and level ratioswere selected to maximize the 2f1-f2 SPL recorded from the externalauditory meatus (Whitehead et al., 1995a, 1995b, 1995c). Two separaterealistic dual radial horn tweeters (Radio Shack, Tandy Corp., Ft Worth,Tex., USA) were used to present the primaries, f2 and f1. The primarieswere acoustically mixed in the external auditory meatus to avoidartifactual distortion. An ER-10B+emissions microphone assembly(Etymotic Research, Elk Grove Village, Ill., USA) was used to captureSPLs in the external auditory meatus. A customized signal presentation,acquisition and analysis program written in LabVIEW version 7.1(National Instruments, Austin, Tex., USA) was used to drive a PCI-4461computer-based DSP board (National Instruments, Austin, Tex., USA). Thisallowed for the delivery of the primaries, synchronous averaging andFourier analysis of 2f1-f2 dB SPLs as a function of f2 frequenciesranging from 3.2 to 63 kHz in 0.1-octave increments. The noise floor wascomputed by averaging SPLs from the external auditory meatus forfrequency bins above and below the 2f1-f2 bin (±3.75 Hz). The measuringmicrophone assembly and the stimulus delivery system were extended to aprobe that was physically and acoustically coupled to each animal'sexternal auditory meatus. A 0.2 cm2 hard-walled cavity that approximatesthe rat's external auditory meatus was used to calibrate the DPOAErecordings. These calibrations were free of artifacts and did notproduce DPOAE SPLs that exceeded the noise floor. A DPOAE is consideredto be present when the SPL exceed the noise floor by at least 3 dB.

Assessment of Neural Function

The CAP was used to access the sensitivity of the auditory branch of theVIIIth craniofacial nerve in the right ear. This procedure is terminal(nonsurvival) and therefore deployed at the end of the study (4 weekspost-noise exposure). The animals were anesthetized with xylazine (13mg/kg, im) and ketamine (87 mg/kg, im) while normal body temperature wasmaintained using a dc heating unit built into the surgical table. Allrecordings were obtained in a double-walled sound-isolation chamber(Industrial Acoustics Inc.). The auditory bulla was approached andopened via a ventrolateral surgical approach. The cochlea was warmedusing a low-voltage high-intensity lamp. A fine Teflon-coatedsilver-wire-recording electrode (A-M Systems, Inc., Carlsborg, Wash.,USA) with an outer-diameter of 0.1 mm was placed on the round windowmembrane while a silver chloride electrode (ground) was inserted intoneck musculature. A speaker-probe assembly was acoustically coupled tothe surgically-resected external auditory meatus. A customized programwritten in LabVIEW 7.1 (National Instruments) was used to drive aSoundMax Integrated Digital Audio board in order to generate and shapestimulus frequency, timing and intensity. Stimulus frequencies wereshaped as a 10 ms burst with ramps of 1 ms on/off-sets. Frequenciesbetween 2 and 40 kHz in approximately ½ octave steps were presentedthrough the probe assembly at a rate of 9.7/s per frequency. Theintensity of the stimulus was adjusted in 1 dB steps until an actionpotential was discernible on a TDS1002 digital oscilloscope (TektronixInc., Beaverton, Oreg., USA). Action potentials were amplified(1000-fold) between 0.1 and 1.0 kHz with a Grass A.C. preamplifier(Model P15, W. Warwick, R.I., USA) and averaged over four sweeps. Neuralsensitivity was tracked by monitoring the N1 action potential from adescending intensity series. The N1 component of the action potentialwas identified based on its shape and latency relative to stimulusonset. The approximate response amplitude of the N1 component at thelowest stimulus needed to stimulate the nerve was 1 mV as measured atthe output of the preamplifier. Neural sensitivity for a particularfrequency was the lowest stimulus intensity in dB SPL needed to elicitan N1 above background noise.

Cytocochleogram

OHCs are among the most sensitive cell types to noise-injury thereforecytocochleograms of missing OHCs were constructed for each animal'sright ear. This was conducted at the end of the study (4 weekspost-noise exposure) on the same animals that received CAP testing.Under high-dose anesthesia (xylazine/ketamine 13/87 mg/kg, im) eachanimal was decapitated and within 60 s cochleae were fixed byround-window perilymphatic perfusion with 1 mL ofperiodate-lysine-paraformaldehyde (PLP). The cochleae were thenpost-fixed for 24 h at 22° C. in PLP. Following fixation the cochlea waswashed with 0.1 M phosphate buffered saline then stained with 2% OsO4 inwater for 2 h and finally washed again with 70% ethanol. The cochlearneurosensory epithelium was micro-dissected in 70% ethanol then mountedin glycerin on microscope slides. A 40× objective mounted on a CarlZeiss upright light microscope was used to visualize and count OHCs.OHCs were counted as present when the cell nucleus could be visualized.The degree of cellular damage to surviving cells was not determined. TheMüller-rat frequency-place map was used to estimate frequency loci as afunction of length along the cochlear spiral (Müller, 1991). This mapreflects the logarithmic-tonotopic scale of a rat's cochlea where highfrequency receptive OHCs are located at the base while low frequencyreceptive OHCs are located at the apex. A cytocochleogram showing thepercentage of OHC loss as a function of distance from the apex of thecochlea was plotted for each animal. The results were then averagedacross each group of subjects for between-group comparisons.

Statistical Analyses

All statistical analyses were conducted with Prism 5 version 5.03(GraphPad Software, Inc., La Jolla, Calif., USA). The DPOAE data wasanalyzed for within-group effects. A 16×4 repeated measures analysis ofvariance (ANOVA) was calculated for DPOAE levels where 16 frequencieswithin 8-24 kHz were compared across four time points (baseline, 1 day,1 week and 4 weeks post-noise exposure) within each group (control,noise-only, CAE-only and CAE+noise). Dunnett's post-hoc analyses wereperformed to determine statistically significant differences betweenDPOAE levels obtained at baseline compared with those obtained atsubsequent time points; 1 day, 1 week and 4 weeks post-noise exposure.The frequency range between 8 and 24 kHz was chosen for statisticalanalysis because it was the largest area affected by the 8 kHz OBN andit is 1½-octave above the center frequency (8 kHz) of the OBN. This highfrequency shift is common among humans and animals exposed to damagingnoise and represent the interaction between the maximum displacement ofthe basilar membrane and the Helmholtz resonance of the externalauditory meatus (Johnstone et al., 1986).

The CAP data were analyzed for between-group effects. Therefore, CAPrecordings of neural sensitivity in dB SPL were subjected to an 11×4two-way ANOVA where 11 frequencies (˜½ octave steps) within 2-40 kHzwere compared across the four treatment groups (control, noise-only,CAE-only and CAE+noise). Lastly, the OHC-count data was also analyzedfor between-group effects. A 29×4 two-way ANOVA was calculated where thepercent of missing OHCs at 29 serial locations (0.31 mm increments alongthe entire epithelium) within 9 mm of the neurosensory epithelium wascompared between the four treatment groups. Data from CAP and OHC-countswere treated with Dunnett's post-hoc testing to determine significantdifferences relative to the control group.

Results

Sensory Function

Sensory function as determined by distortion product otoacousticemission (DPOAE) revealed that CAE treatment preserved outer hair cell(OHC) activity following noise exposure. All groups started and endedthe study at the same time therefore DPOAE measurements fromvehicle-only and CAE-only groups followed the same time schedule as thenoise exposure groups. FIG. 1A shows DPOAE measurements for thevehicle-only group (control). These measurements reveal that DPOAElevels exhibited little variability across time-points. This smallvariability is representative of normal physiologically active OHCs(Wagner et al., 2008). FIG. 1B shows DPOAE levels for the noise treatedgroup. Note that FIG. 1B shows a significant loss in DPOAE levels at 1day following noise exposure. At 1 week following noise exposure DPOAElevels of apical (low frequency: 3-7 kHz) and basal (high frequency:25-34 kHz) components showed marked recovery. However, the middlefrequency components (8-24 kHz) and the highest frequency components(frequencies greater than 34 kHz) failed to recover to the same extentas the other frequency components. At 4 weeks post-noise exposure therewas a slight improvement in DPOAE levels for the middle frequencycomponents, however, these DPOAE components and the highest frequencycomponents are still significantly depressed relative to that atbaseline. These remaining depressions in DPOAE levels are consistentwith a permanent sensory impairment. The combined results suggest thatthe noise exposure induced permanent loss of DPOAE levels 1-4 weeksfollowing noise-treatment.

FIG. 1C shows that CAE treatment alone does not adversely affect OHCfunction and similar to the control group, DPOAE levels exhibited littlevariability between time-points. FIG. 1D shows DPOAE levels for theCAE+noise treated group. Note the significant loss in DPOAE levels at 1day following noise exposure. At 1 week following noise exposure DPOAElevels across-frequencies showed marked recovery. Interestingly, thisrecovery was almost complete at only 1 week following noise exposurewhich indicates significant preservation of OHC function. Additionally,at 4 weeks post-noise exposure DPOAE levels across frequencies furtherimproved to approximate DPOAE levels recorded at baseline. The combinedresults suggest that CAE treatment facilitated almost complete recoveryof OHC function following noise injury.

Statistical analyses were conducted on DPOAE levels. Table 2 provides asummary of the F-ratios from several two-way repeated measures ANOVAswhere time (baseline, 1 day, 1 week and 4 weeks) and frequency (8-24kHz) served as main effects for the four groups (control, noise, CAE,CAE+noise). For each group Dunnett's post-hoc testing was used tocompare the mean DPOAE levels at baseline to the other three timepoints. FIG. 1 shows that the noise only group exhibited the largestDPOAE amplitude shifts (Dunnett p<0.05) between baseline and the otherthree time points.

TABLE 2 Results of two-way repeated measures analysis of variance onDPOAE levels. F-values Source df Control Noise CAE CAE + noise Time (T)3 12.33^(a) 128.3^(a) 11.82^(a) 114.9^(a) Frequency (F) 15 2.386^(a)0.621 2.863^(a) 0.3557 T × F 45 0.2471 1.514^(b) 3.072^(a) 1.576^(b)MS_(ERROR) (28.49) (77.34) (24.67) (44.93) The F-ratios are shown forseparate analyses on DPOAE levels from the four treatment groups(control group, noise-only group, CAE-only group and CAE + noise group).Significant main effects of time (baseline, 1-day, 1-week and 4-weekspost-noise exposure) and frequency (8-24 kHz) and the significant time ×frequency interactions are indicated. ^(a)p < 0.01. ^(b)p < 0.05.

Neural Function

Compound action potential (CAP) recordings suggested that CAE treatmentlimited neural impairment following noise exposure. FIG. 3 revealsneural sensitivity within a frequency range of 2-40 kHz for the fourtreatment groups at 4 weeks post-noise exposure. Note that the noiseexposure produced a significant loss in neural sensitivity while the CAEtreatment provided significant protection (ANOVA main effect oftreatment; F3,264=65.22, p<0.01). In the noise-only group, neuralsensitivity was significantly impaired between 8.0 and 40.0 kHz wherethe average sensitivity loss was 28 dB relative to untreated controlsubjects (Dunnett's post-hoc contrast; p<0.05). For the CAE treatedgroup, neural sensitivity was slightly elevated between 8.0 and 40.0 kHzwhere the average sensitivity loss was only 7 dB relative to untreatedcontrol subjects (Dunnett's post-hoc contrast; p>0.05). This slightshift of 7 dB indicates that systemic CAE treatment affects inner earneurons possibly by potentiating endogenous mechanisms (see Discussionsection). In the CAE+noise group neural sensitivity was mildly impairedbetween 8.0 and 40.0 kHz where the average sensitivity loss was 13 dBrelative to untreated control subjects (Dunnett's post-hoc contrast;p<0.05). This mild impairment suggests that the noise treatment had adamaging effect in this group, but the damage was less than that of thenoise-only group.

Cytocochleogram

Cytocochleograms of OHC counts from each group of animals revealed thatthe noise exposure induced cellular loss, but CAE treatment limited themagnitude of the loss. FIG. 2 reveals OHC loss (for rows 1-3) along thelength of the cochlear neurosensory epithelium for the differenttreatment groups (control, noise only, CAE-only and CAE+noise) at a 4week post-noise exposure survival time. There were statisticallysignificant differences between the four treatment groups (ANOVA maineffect of treatment; F3,232=21.45, p<0.01). Cell loss as a function ofdistance along the neurosensory epithelium of the control group provideda reference for determining the effect of individual treatment(noise-only, CAE-only or CAE+noise). In the noise-only group cell losswas significant toward the basal end of the neurosensory epitheliarelative to that of the untreated control subjects (Dunnett's post-hoccontrast; p<0.01). This suggests that the noise exposure producedpermanent cell loss which was more prominent at the basal coil(high-frequency responsive area). For the CAE treated group cell losswas similar to that of untreated control subjects (Dunnett's post-hoccontrast; p>0.05). This reveals that the CAE treatment does notadversely affect the OHCs. In the CAE+noise group there was a mildincrease in cell loss relative to untreated control subjects (Dunnett'spost-hoc contrast; p<0.05). This mild loss suggests that the noisetreatment had a damaging effect in this group, but the damage was lessthan that of the noise-only group.

Discussion

This study provides the first demonstration that treatment with CAE ofUT may augment recovery of sensory and neural functions following noiseinjury. Exposure to an 8 kHz octave band of noise (OBN) at 105 dB SPLfor four hours significantly reduced DPOAE levels in all animals at 1day post-exposure. However, at 1 week post-exposure the CAE treatedanimals showed almost complete recovery of DPOAE levels while theanimals which received only noise failed to show significant recovery.At 4 weeks post-noise exposure the CAE treated animals exhibitedadditional recovery in DPOAE levels while the animals which receivedonly noise continued to experience reduced DPOAE levels. These findingssuggest that CAEs may enhance the recovery of OHC function from bothtemporary (1 week) and permanent (4 weeks) noise-injury. Indeed,cytocochleograms of the percent of missing OHCs revealed that CAEtreated animals suffered from less OHC loss compared to animals whichreceived only noise. The functional and structural preservation observedfor OHCs was consistent with round-window recordings of actionpotentials. Here, CAE treatment prevented significant impairment inneural sensitivity following noise exposure. The combined results implyan otoprotective role for CAE in noise-injury.

REFERENCES FOR EXAMPLE 1

-   Akesson, C., Lindgren, H., Pero, R. W., Leanderson, T., Ivars, F.,    2003a. An extract of Uncaria tomentosa inhibiting cell division and    NF-kappa B activity without inducing cell death. Int.    Immunopharmacol. 3, 1889-900.-   Akesson, C., Lindgren, H., Pero, R. W., Leanderson, T., Ivars,    F., 2005. Quinic acid is a biologically active component of the    Uncaria tomentosa extract C-Med 100. Int Immunopharmacol. 5,    219-229.-   Akesson, C., Pero, R. W., Ivars, F., 2003b. C-Med 100, a hot water    extract of Uncaria tomentosa, prolongs lymphocyte survival in vivo.    Phytomedicine. 10, 23-33.-   Bacher, N., Tiefenthaler, M., Sturm, S., Stuppner, H.,    Ausserlechner, M. J., Kofler, R., Konwalinka, G., 2006. Oxindole    alkaloids from Uncaria tomentosa induce apoptosis in proliferating,    G0/G1-arrested and bc1-2-expressing acute lymphoblastic leukaemia    cells. Br J Haematol. 132, 615-622.-   Bartos, J., 1980. Colorimetric determination of organic compounds by    formation of hydroxamic acids. Talanta. 27, 583-590.-   Basta, D., Tzschentke, B., Ernst, A., 2005. Noise-induced cell death    in the mouse medial geniculate body and primary auditory cortex.    Neurosci Lett. 381, 199-204.-   Belkaid, A., Currie, J. C., Desgagnés, J., Annabi, B., 2006. The    chemopreventive properties of chlorogenic acid reveal a potential    new role for the microsomal glucose-6-phosphate translocase in brain    tumor progression. Cancer Cell Int. 6, 7.-   Chen, G. D., Fechter, L. D., 2003. The relationship between    noise-induced hearing loss and hair cell loss in rats. Hear Res.    177, 81-90.-   Chung, J. S., Lee, S. B., Park, S. H., Kang, S. T., Na, A. R.,    Chang, T. S., Kim, H. J., Yoo, Y. D., 2009. Mitochondrial reactive    oxygen species originating from Romo1 exert an important role in    normal cell cycle progression by regulating p27(Kip1) expression.    Free Radic Res. 43, 729-737.-   Coling, D. E., Yu, K. C., Somand, D., Satar, B., Bai, U., Huang, T.    T., Seidman, M. D., Epstein, C. J., Mhatre, A. N., Lalwani, A.    K., 2003. Effect of SOD1 overexpression on age- and noise-related    hearing loss. Free Radic Biol Med. 34, 873-880.-   Desmarchelier, C., Mongelli, E., Coussio, J., Ciccia, G., 1997.    Evaluation of the in vitro antioxidant activity in extracts of    Uncaria tomentosa (Willd.) DC. Phytotherapy Research. 11, 254-256.-   Endo, T., Nakagawa, T., Iguchi, F., Kita, T., Okano, T., Sha, S H.,    Schacht, J., Shiga, A., Kim, T. S., Ito, J., 2005. Elevation of    superoxide dismutase increases acoustic trauma from noise exposure.    Free Radic Biol Med. 38, 492-498.-   Fujioka, M., Kanzaki, S., Okano, H. J., Masuda, M., Ogawa, K.,    Okano, H., 2006. Proinflammatory cytokines expression in    noise-induced damaged cochlea. J Neurosci Res. 83, 575-583.-   Gonçalves, C., Dinis, T., Batista, M. T., 2005. Antioxidant    properties of proanthocyanidins of Uncaria tomentosa bark decoction:    a mechanism for anti-inflammatory activity. Phytochemistry. 66,    89-98.-   Gurrola-Díaz, C. M., García-López, P. M., Gulewicz, K., Pilarski,    R., Dihlmann, S., 2010 Inhibitory mechanisms of two Uncaria    tomentosa extracts affecting the Wnt-signaling pathway.    Phytomedicine. doi:10.1016-   Guthrie, O. W., Carrero-Martínez, F. A., 2010. Real-time    quantification of Xeroderma pigmentosum mRNA from the mammalian    cochlea. Ear Hear. 31, 714-721.-   Guthrie, O. W., 2009. DNA repair proteins and telomerase reverse    transcriptase in the cochlear lateral wall of cisplatin-treated    rats. J Chemother. 21, 74-79.-   Hakuba, N., Koga, K., Gyo, K., Usami, S. I., Tanaka, K., 2000.    Exacerbation of noise-induced hearing loss in mice lacking the    glutamate transporter GLAST. J Neurosci. 20, 8750-8753.-   Han, W., Shi, X., Nuttall, A. L., 2006. AIF and endoG translocation    in noise exposure induced hair cell death. Hear Res. 211, 85-95.-   Hirose, K., Discolo, C. M., Keasler, J. R., Ransohoff, R., 2005.    Mononuclear phagocytes migrate into the murine cochlea after    acoustic trauma. J Comp Neurol. 489, 180-194.-   Hu, B. H., Henderson, D., Nicotera, T. M., 2006. Extremely rapid    induction of outer hair cell apoptosis in the chinchilla cochlea    following exposure to impulse noise. Hear Res. 211, 16-25.-   Johnstone, B. M., Patuzzi, R., Yates, G. K., 1986. Basilar membrane    measurements and the travelling wave. Hear Res. 22, 147-153.-   Keplinger, K., Laus, G., Wurm, M., Dierich, M. P., Teppner,    H., 1999. Uncaria tomentosa (Willd.) DC.—ethnomedicinal use and new    pharmacological, toxicological and botanical results. J    Ethnopharmacol. 64, 23-34.-   Lamm, S., Sheng, Y., Pero, R. W., 2001. Persistent response to    pneumococcal vaccine in individuals supplemented with a novel water    soluble extract of Uncaria tomentosa, C-Med-100. Phytomedicine. 8,    267-274.-   Laus, G., 2004. Advances in chemistry and bioactivity of the genus    Uncaria. Phytother Res. 18, 259-274.-   Lemaire, I., Assinewe, V., Cano, P., Awang, D. V., Amason, J.    T., 1999. Stimulation of interleukin-1 and -6 production in alveolar    macrophages by the neotropical liana, Uncaria tomentosa (uña de    gato). Ethnopharmacol. 64, 109-115.-   Lorito, G., Giordano, P., Prosser, S., Martini, A., Hatzopoulos,    S., 2006. Noise-induced hearing loss: a study on the pharmacological    protection in the Sprague Dawley rat with N-acetyl-cysteine. Acta    Otorhinolaryngol Ital. 26, 133-139.-   Mammone, T., Akesson, C., Gan, D., Giampapa, V., Pero, R. W., 2006.    A water soluble extract from Uncaria tomentosa (Cat's Claw) is a    potent enhancer of DNA repair in primary organ cultures of human    skin. Phytother Res. 20, 178-183.-   Masuda, M., Nagashima, R., Kanzaki, S., Fujioka, M., Ogita, K.,    Ogawa, K., 2006. Nuclear factor-kappa B nuclear translocation in the    cochlea of mice following acoustic overstimulation. Brain Res. 1068,    237-247.-   McFadden, S. L., Ohlemiller, K. K., Ding, D., Shero, M., Salvi, R.    J., 2001. The Influence of Superoxide Dismutase and Glutathione    Peroxidase Deficiencies on Noise-Induced Hearing Loss in Mice. Noise    Health. 3, 49-64.-   Minami, S. B., Yamashita, D., Ogawa, K., Schacht, J., Miller, J.    M., 2007. Creatine and tempol attenuate noise-induced hearing loss.    Brain Res. 1148, 83-89.-   Mohr, P. E., Feldman, J. J., Dunbar, J. L., McConkey-Robbins, A.,    Niparko, J. K., Rittenhouse, R. K., Skinner, M. W., 2000. The    societal costs of severe to profound hearing loss in the United    States. Int J Technol Assess Health Care. 16, 1120-1135.-   Müller, M., 1991. Frequency representation in the rat cochlea. Hear    Res. 51, 247-254.-   Nicotera, T. M., Hu, B. H., Henderson, D., 2003. The caspase pathway    in noise-induced apoptosis of the chinchilla cochlea. J Assoc Res    Otolaryngol. 4, 466-477.-   Ohlemiller, K. K., 2003. Oxidative cochlear injury and the    limitations of antioxidant therapy. Semin Hear. 24, 123-134.-   Ohlemiller, K. K., 2008. Recent findings and emerging questions in    cochlear noise injury. Hear Res. 245, 5-17.-   Ohlemiller, K. K., Wright, J. S., Dugan, L. L., 1999. Early    elevation of cochlear reactive oxygen species following noise    exposure. Audiol Neurootol. 4, 229-236.-   Pero, R. W., Amiri, A., Sheng, Y., Welther, M., Rich, M., 2005.    Formulation and in vitro/in vivo evaluation of combining DNA repair    and immune enhancing nutritional supplements. Phytomedicine. 2005,    12, 255-263.-   Pero, R. W., Giampapa, V., Vojdani, A., 2002. Comparison of a broad    spectrum anti-aging nutritional supplement with and without the    action of a DNA repair enhancing cat's claw extract. J Anti Aging    Med. 5, 345-355.-   Pero, R. W., 2010. Health consequences of catabolic synthesis of    hippuric acid in humans. Curr Clin Pharmacol. 5, 67-73.-   Pero, R. W., Lund, H., 2010. Dietary quinic acid supplied as the    nutritional supplement AIO+AC-11® leads to induction of micromolar    levels of nicotinamide and tryptophan in the urine. Phytother Res.    doi: 10.1002/ptr.3348.-   Pero, R. W., Lund, H., Leanderson, T., 2009. Antioxidant metabolism    induced by quinic acid. Increased urinary excretion of tryptophan    and nicotinamide. Phytother Res. 23, 335-346.-   Pienkowski, M., Eggermont, J. J., 2010. Intermittent exposure with    moderate-level sound impairs central auditory function of mature    animals without concomitant hearing loss. Hear Res. 261, 30-35.-   Pilarski, R., Zieliński, H., Ciesiolka, D., Gulewicz, K., 2005.    Antioxidant activity of ethanolic and aqueous extracts of Uncaria    tomentosa (Willd.) DC. J Ethnopharmacol. 104, 18-23.-   Sandoval, M., Okuhama, N. N., Zhang, X. J., Condezo, L. A., Lao, J.,    Angeles, F. M., Musah, R. A., Bobrowski, P., Miller, M. J., 2002.    Anti-inflammatory and antioxidant activities of cat's claw (Uncaria    tomentosa and Uncaria guianensis) are independent of their alkaloid    content. Phytomedicine. 9, 325-337.-   Satou, K., Hori, M., Kawai, K., Kasai, H., Harashima, H., Kamiya,    H., 2009. Involvement of specialized DNA polymerases in mutagenesis    by 8-hydroxy-dGTP in human cells. DNA Repair. 8, 637-642.-   Shen, H., Zhang, B., Shin, J. H., Lei, D., Du, Y., Gao, X., Wang,    Q., Ohlemiller, K. K., Piccirillo, J., Bao, J., 2007. Prophylactic    and therapeutic functions of T-type calcium blockers against    noise-induced hearing loss. Hear Res. 226, 52-60.-   Sheng, Y., Akesson, C., Holmgren, K., Bryngelsson, C., Giamapa, V.,    Pero, R. W., 2005. An active ingredient of Cat's Claw water extracts    identification and efficacy of quinic acid. J Ethnopharmacol. 96,    577-584.-   Sheng, Y., Bryngelsson, C., Pero, R. W., 2000a. Enhanced DNA repair,    immune function and reduced toxicity of C-MED-100, a novel aqueous    extract from Uncaria tomentosa. J Ethnopharmacol. 69, 115-126.-   Sheng, Y., Li, L., Holmgren, K., Pero, R. W., 2001. DNA repair    enhancement of aqueous extracts of Uncaria tomentosa in a human    volunteer study. Phytomedicine. 8, 275-282.-   Sheng, Y., Pero, R. W., Wagner, H., 2000b. Treatment of    chemotherapy-induced leukopenia in a rat model with aqueous extract    from Uncaria tomentosa. Phytomedicine. 7, 137-143.-   Schildkraut, E., Miller, C. A., Nickoloff, J. A., 2005. Gene    conversion and deletion frequencies during double-strand break    repair in human cells are controlled by the distance between direct    repeats. Nucleic Acids Res. 33, 1574-1580.-   Van Campen, L. E., Murphy, W. J., Franks, J. R., Mathias, P. I.,    Toraason, M. A., 2002. Oxidative DNA damage is associated with    intense noise exposure in the rat. Hear Res. 164, 29-38.-   Wang, J., Ruel, J., Ladrech, S., Bonny, C., van de Water, T. R.,    Puel, J. L., 2007 Inhibition of the c-Jun N-terminal kinase-mediated    mitochondrial cell death pathway restores auditory function in    sound-exposed animals. Mol Pharmacol. 71, 654-666.-   Wagner, H., Kreutzkamp, B., Jurcic, K., 1985. The alkaloids of    Uncaria tomentosa and their phagocytosis-stimulating action. Planta    Med. 5, 419-423.-   Wagner, W., Heppelmann, G., Vonthein, R., Zenner, H. P., 2008.    Test-retest repeatability of distortion product otoacoustic    emissions. Ear Hear. 29, 378-391.

Whitehead, M. L., Stagner, B. B., Lonsbury-Martin, B. L., Martin, G. K.,1995a. Effects of ear-canal standing waves on measurements ofdistortion-product otoacoustic emissions. J Acoust Soc Am. 98,3200-3214.

-   Whitehead, M. L., Stagner, B. B., McCoy, M. J., Lonsbury-Martin, B.    L., Martin, G. K., 1995b. Dependence of distortion-product    otoacoustic emissions on primary levels in normal and impaired    ears. II. Asymmetry in L1, L2 space. J Acoust Soc Am. 97, 2359-2377.-   Whitehead, M. L., McCoy, M. J., Lonsbury-Martin, B. L., Martin, G.    K., 1995c. Dependence of distortion-product otoacoustic emissions on    primary levels in normal and impaired ears. I. Effects of decreasing    L2 below L1. J Acoust Soc Am. 97, 2346-2358.-   Yamane, H., Nakai, Y., Takayama, M., Iguchi, H., Nakagawa, T.,    Kojima, A., 1995. Appearance of free radicals in the guinea pig    inner ear after noise-induced acoustic trauma. Eur Arch    Otorhinolaryngol. 252, 504-508.-   Yamashita, D., Jiang, H. Y., Schacht, J., Miller, J. M., 2004a.    Delayed production of free radicals following noise exposure. Brain    Res. 1019, 201-209.-   Yamashita, D., Miller, J. M., Jiang, H. Y., Minami, S. B., Schacht,    J., 2004b AIF and EndoG in noise-induced hearing loss. Neuroreport.    15, 2719-2722.-   Yang, W. P., Henderson, D., Hu, B. H., Nicotera, T. M., 2004.    Quantitative analysis of apoptotic and necrotic outer hair cells    after exposure to different levels of continuous noise. Hear Res.    196, 69-76.

Example 2

In response to stress, spiral ganglion neurons may remodel intracellularpools of DNA repair proteins. This hypothesis was addressed bydetermining the intracellular location of three classic DNA excisionrepair proteins (XPA, CSA and XPC) within the neurons under normalconditions, 1 day after noise stress (105 dB/4 hours) and following DNArepair adjuvant therapy with carboxy alkyl esters (CAEs; 160 mg/kg/28days). Under normal conditions three intracellular compartments wereenriched with at least one repair protein. These intracellularcompartments were designated nuclear, cytoplasmic and perinuclear. Afterthe noise stress each repair protein aggregated in the cytoplasm. AfterCAE therapy each intracellular compartment was enriched with the threeDNA repair proteins. Combining noise stress with CAE therapy resulted inthe enrichment of at least two repair proteins in each intracellularcompartment. The combined results suggest that in response to noisestress and/or otoprotective therapy, spiral ganglion neurons mayselectively remodel compartmentalized DNA repair proteins.

Methods

Animal Care and Use

Experiments were conducted on male Long-Evans rats (250-300 g at 2months old) that were purchased from Harlan Laboratories, Inc.(Livermore, Calif., USA). The animals were housed in pairs inenvironmentally enriched cages in a rat vivarium (21°±1° C.; 12-hourlight/dark cycle). A total of 36 animals served as subjects. Except forthe control group (N=10); the animals were either exposed to noise(N=10), treated with CAE (N=8) or co-treated with CAE+noise (N=8). Allexperimental protocols were approved by the Institutional Animal Careand Use Committee (IACUC) at the Loma Linda VA Medical Center. The IACUCapproval process certifies that all protocols are in compliance with thePublic Health Service (PHS) Policy on Humane Care and Use of LaboratoryAnimals and the Animal Welfare Act.

CAE Treatment

CAE treatment is known to improve DNA repair capacity among humans andrats [27],[29]. Furthermore, previous research has shown that Long-Evansrats gavaged with CAE (160 mg/kg) for 28 days exhibited protection fromnoise induced hearing loss [33]. Therefore this CAE treatment regimenwas employed. Briefly, all animals had free access to water and theirdiet (ad libitum) consisted of standard non-purified Teklad 7001 ratpellets (Teklad Diets, Madison Wis., USA). This diet was supplementedwith CAE for animals in the CAE groups. CAE was obtained from OptigenexInc. (Hoboken, N.J., USA) in powdered form (AC-11C)) and then dissolvedin double-distilled water at a concentration of 160 mg/ml as describedpreviously [27],[33]. A 20-gauge animal feeding stainless steel needlewas used to intubate alert animals in order to administer CAE (160mg/kg). Fresh solutions of CAE were prepared each day and administeredvia gastric intubation for 28 consecutive days. On the 29th day, halfthe animals were treated with noise.

Noise Exposure

Long-Evans rats exposed to an 8 kHz octave band of noise (OBN) at 105 dBSPL for 4 hours are known to exhibit permanent hearing loss [34],[35].Therefore, this noise exposure was used in the current studies and theprotocol has been described previously [33]. Briefly, awake and alertanimals were placed in a small wire-cloth enclosure (15×13×11 cm) withina reverberant 40-L chamber. An HCA1000A Parasound Amplifier (ParasoundProducts, Inc., San Francisco, Calif., USA) was used to drive speakerslocated approximately 5 cm above the wire-cloth enclosure. Soundpressure levels measured at the rats' pinnae were 105 dBlin SPL in theoctave band centered around 8 kHz. These sound pressure measurementswere made using an OB-300 Quest Type-1 Sound Pressure Meter with ⅓octave filter set (Quest Electronics, Oconomowoc, Wis., USA). Noiseexposed animals were subsequently used for immunohistochemical studiesas described below.

Immunohistochemistry

Animal and Tissue Preparation

One day after the noise exposure, five animals from the control andnoise groups and three animals from the CAE and CAE+noise groups (totalof 16 rats) were anesthetized with ketamine/xylazine (87/13 mg/kg, im).After a negative response to a paw pinch, the animals were euthanizedvia transcardial perfusion with phosphate-buffered saline (PBS; 10 mM,pH 7.4) followed by periodate-lysine-paraformaldehyde fixation [36]. Theanimals were then decapitated and the epidermis surgically removed. Thelower mandible was then dissected and the bulla was opened to allow foradditional fixation. The specimens were then post-fixed in 4%paraformaldehyde for at least 24 hrs at 22° C. They were thendecalcified in 10% formic acid and neutralized in 5% sodium sulfite.This procedure has been described previously [6,11,36] and includedincubating the specimens in fresh formic acid every two days at 22° C.Decalcification was monitored every two days by removing 2 ml of theused formic acid and combining this with 1 ml of 5% ammonium hydroxideand 1 ml of 5% ammonium oxalate. End-point decalcification was achievedwhen the formic acid-ammonium hydroxide/oxalate reaction failed toproduce visible white precipitates in three consecutive attempts over 1week. The specimens were then neutralized overnight by incubating in 5%sodium sulfite at 22° C. then rinsed for at least eight hours in dH₂Oprior to trimming and paraffin embedding. Paraffin embedded blocks weresectioned horizontally with a Leica RM2255 rotary microtome (LeicaMicrosystems Inc., Bannockburn, Ill., USA) at 5 or 8 μm and mounted onsubbed slides. Liver tissues were simultaneously harvested, post-fixed,paraffin embedded, sectioned and mounted on subbed slides. The sectionswere then stored at 22° C. prior to immunolabeling.

Immunolabeling

Tissue sections were de-paraffinized then incubated in 30% H₂O₂ for 10min at 22° C. They were then heated to 90-98° C. in a low pH (0.80-3.06)sodium citrate-citric acid buffer for 20 min (antigen retrieval).Afterwards, the sections were rinsed thoroughly with PBS (pH 7.4). Theywere then pre-treated with a blocking solution of normal horse or goatserum, 10% Triton X-100 and 2% bovine serum albumin (BSA; Sigma, St.Louis, Mo., USA) in PBS for 1 hour. The primary antibodies were dilutedin the blocking solution at a 1:100 concentration. The primaryantibodies are commercially available and included anti-XPC (sc-22535),anti-XPA (sc-853) and anti-CSA (sc-25369) (Santa Cruz Biotechnology,Inc., Santa Cruz, Calif., USA). These primary antibodies have beencharacterized previously through pre-absorption experiments,immunohistochemistry and Western blots [36],[37],[12]. Nevertheless,negative control experiments were conducted such that the sections wereincubated with blocking serum instead of the primary antibodies (FIG.5). After 48 hr incubation at 0° C. with the primary antibodies (orblocking serum for negative control sections) the sections were rinsedwith PBS. They were then treated with biotinylated anti-goat oranti-rabbit secondary antibodies (Vector Laboratories, Temecula, Calif.,USA) diluted 1:100 in PBS+2% BSA for 24 hours at 0° C. The sections werethen rinsed in PBS (pH 7.4) and incubated with preformedavidin-biotin-peroxidase complexes (Vectastain ABC reagent; VectorLaboratories, Inc., Burlingame, Calif., USA) for one hour, rinsed againwith PBS and then treated with a solution of Trizma pre-set crystals(1.58 g; Sigma-Aldrich, St. Louis, Mo., USA). Afterwards the sectionswere washed in PBS (pH 7.4) and stained for 10 min with3,3′-diaminobenzidine tetrahydrochloride. These stained sections werethen dehydrated in a graded series of ethyl alcohol then cleared withxylene and cover slipped in Shandon-Mount (Anatomical Pathology,Pittsburgh, Pa., USA).

Data Analysis

Spiral ganglion cells were counted by three researchers who were blindto the experimental conditions. For each animal, 80 μm (control andnoise groups) or 45 μm (CAE and CAE+noise groups) of mid-modiolarcochlear sections were analyzed under oil immersion at 100×magnification. The number of spiral ganglion cells showing cytoplasmic,diffuse, nuclear or perinuclear immunoreactivity was determined for eachof the three proteins (XPC, XPA and CSA). Profiles of spiral ganglioncells were detected throughout the entire thickness of the sections andonly neurons with a well-defined soma and nucleus was included in thecell counts. Image-Pro® plus version 6.3 (Media Cybernetics Inc.,Bethesda, Md., USA) for Windows™ was used for recording of 1-pixel widelinescans. These linescans were recorded along the central axis of thecell bodies of neurons to objectively verify the subcellularlocalization patterns (FIGS. 6C, F, I and L). Statistical differenceswere determined with analysis of variance (ANOVA) followed byTukey-Kramer post-hoc testing. Within group analyses were conducted,therefore within a particular group the number of cells that exhibiteach pattern was compared to determine significant differences betweenthe patterns. Additionally, between group comparisons were conducted todetermine differences as a function of treatment conditions.

Results

Controls

DNA repair activity in the rat liver is among the highest of all themajor organs such as heart, brain, lung, spleen and muscle [38].Furthermore, the mRNA of several types of DNA repair genes including XPCand XPA has previously been purified from the liver [39]. Additionally,recent immunohistochemical experiments have confirmed the expression ofDNA repair proteins such as XPC and XPA within rat hepatocytes [40].Therefore in the current study, rat hepatocytes served as positivecontrols for the immunohistochemical experiments. FIG. 5A is a Nomarskimicrograph that reveals XPC immunoreactivity within hepatocytes. Thereaction products are prominent and granular to homogenous inappearance. This immunoreactivity was representative of that observedfor XPA and CSA within hepatocytes. Omitting only the antibodies fromthe immunohistochemical procedure (negative control) yielded no reactionproducts in hepatocytes or spiral ganglion neurons (FIG. 5B-5D).

Intracellular Distribution Patterns

Spiral ganglion neurons were found to compartmentalize the repairproteins in their cytoplasm, nucleoplasm or at perinuclear loci. Thiscompartmentalization was manifested in four distinct intracellulardistribution patterns designated as diffuse, cytoplasmic, nuclear andperinuclear. FIG. 6 reveals each of these patterns for the XPC protein,which is representative of the other DNA repair proteins. FIGS. 6A-6Bare high resolution photomicrographs of the diffuse pattern. Theimmunoreactivity is diffused throughout the soma and there is no cleardistinction between the nucleoplasm and the cytoplasm. This pattern canbe objectively profiled with linescans across the diameter of the somaas demonstrated in FIG. 6C. FIGS. 6D-6E are representative highresolution photomicrographs of the cytoplasmic localization pattern.This particular localization pattern can be objectively profiled withlinescans across the diameter of the soma as demonstrated in FIG. 6F.FIGS. 6G-6H reveals the nuclear localization pattern whereimmunoreactivity is predominantly localized in the nucleoplasm. Thispattern shows a distinct linescan profile (FIG. 61). FIGS. 6J-6Killustrate the perinuclear localization pattern which also produces aunique linescan signature (FIG. 6L).

Cell Counts

FIG. 7 is an illustration of spiral ganglion cell counts as a functionof distribution pattern from the control group. Note that all threeintracellular compartments (cytoplasmic, nuclear and perinuclear) wereenriched with at least one protein. For instance, the XPC protein waspreferentially compartmentalized in the cytoplasm while the XPA proteinwas diffused (diffuse pattern) throughout the cytoplasm and the nucleus.Furthermore, the CSA protein was preferentially localized in thecytoplasm and at perinuclear loci. This heterogeneous distribution wasfurther supported by statistical analyses conducted on the number ofpatterns derived from individual proteins. A one-way repeated measureANOVA revealed significant differences between the intracellulardistribution patterns for the XPC (p<0.001), XPA (p<0.001) and CSA(p<0.001) proteins. For instance, there was a gradient in thedistribution of XPC and Tukey-Kramer pairwise comparisons revealed thatthe cytoplasmic distribution pattern was significantly higher than theother patterns (p<0.05). XPA positive cells showed significantcytoplasmic, diffuse and nuclear patterns compared to the perinuclearpattern but the diffuse pattern was the most dominant (Tukey-Kramerpairwise contrasts; p<0.05). For the CSA protein, the cytoplasmic andperinuclear patterns were significantly higher than the other patterns(Tukey-Kramer pairwise contrasts; p<0.05).

FIG. 8 is an illustration of spiral ganglion cell counts as a functionof distribution pattern following the noise exposure. Note that allthree proteins (XPC, XPA and CSA) were preferentially compartmentalizedin the cytoplasm. This uniform cytoplasmic response is in contrast withthat of the control condition where all compartments were enriched withat least one repair protein. A one-way repeated measure ANOVA revealedsignificant differences between the intracellular distribution patternsfor the XPC (p<0.001), XPA (p<0.001) and CSA (p<0.001) proteins. Forinstance, the cell counts as a function of distribution pattern can bedescribed as a gradient where cytoplasmic location is the mostsignificant (Tukey-Kramer pairwise contrasts; p<0.05).

FIG. 9 is an illustration of spiral ganglion cell counts as a functionof distribution pattern following CAE treatment. Gradients in the cellcounts for all three proteins could be detected. However, unlike noiseexposure there was no preference for a single distribution pattern orintracellular compartment. For instance, a one-way repeated measuresANOVA revealed no significant differences between the intracellulardistribution patterns for the XPC (p>0.05), XPA (p>0.05) and CSA(p>0.05) proteins. Therefore, after CAE treatment no one distributionpattern emerged as more significant that the other patterns for a givenprotein (Tukey-Kramer pairwise contrasts; p>0.05).

FIG. 10 is an illustration of spiral ganglion cell counts as a functionof distribution pattern following co-treatment with CAE and noise. Theresults appear as a merger between the results from the control and theCAE-only groups. For instance, similar to the CAE-only group, gradientsin the cell counts for all three proteins could be detected. However, aone-way repeated measure ANOVA revealed no significant differencesbetween the intracellular localization patterns for the XPC (p>0.05) andXPA (p>0.05) proteins. For each of these proteins no one distributionpattern emerged as more significant than the other patterns(Tukey-Kramer pairwise contrasts; p>0.05). The data for the CSA proteinwas similar to that of the control group. A one-way repeated measureANOVA revealed significant differences between the intracellulardistribution patterns (p<0.05). For instance, the cytoplasmic andperinuclear patterns were significantly higher than the other patterns(Tukey-Kramer pairwise contrasts; p<0.05).

FIG. 11 reveals the effect of the four experimental conditions (control,noise exposed, CAE-only and CAE+noise) on the four distribution patterns(cytoplasmic, diffused, nuclear and perinuclear). One-way ANOVA testingon the mean number of XPC positive cells from each group revealed nosignificant (p>0.05) differences across distribution patterns. Thisimplies that treatment condition does not affect the number of cellsimmunopositive for XPC regardless of subcellular distribution. However,ANOVA testing on the mean number of XPA positive cells from each grouprevealed significant (p<0.01) differences that were dependent on thedistribution patterns. For instance, the cytoplasmic distributionpattern showed a significant (Tukey-Kramer pairwise contrasts; p<0.05)difference only between noise exposure and CAE-only treatment. Thediffused distribution pattern exhibited significant differences betweenthe groups (ANOVA; p<0.01). For instance, significant differences wereevident between control and noise exposure (Tukey-Kramer pairwisecontrasts; p<0.01) and between control and CAE-only treatment(Tukey-Kramer pairwise contrasts; p<0.05). Interestingly, the nucleardistribution pattern did not exhibit significant (ANOVA: p>0.05)differences between the groups. In contract, the perinucleardistribution pattern evidenced significant (ANOVA: p<0.05) differencesbetween the groups and post host testing revealed that this differencewas only evident between the control and CAE+noise groups (Tukey-Kramerpairwise contrasts; p<0.05). The combined results for XPA suggest thatthe number of cells immunopositive for XPA is affected by the treatmentconditions and subcellular distribution.

Statistical testing on the mean number of CSA positive cells from eachgroup revealed significant (ANOVA; p<0.01) differences in the number ofcell exhibiting a cytoplasmic distribution pattern. For instance,Tukey-Kramer pairwise testing revealed significant difference betweencontrol and CAE (p<0.01), control and CAE+noise (p<0.01), noise and CAE(p<0.05), noise and CAE+noise (p<0.01) and CAE and CAE+noise (p<0.05).The diffused distribution pattern exhibited significant differencesbetween the groups (ANOVA; p<0.01). For instance, significantdifferences were evident between control and CAE treatment (Tukey-Kramerpairwise contrasts; p<0.01) and between noise and CAE-only treatment(Tukey-Kramer pairwise contrasts; p<0.05). There were no significantdifferences between the groups for the other distribution patterns(Tukey-Kramer pairwise contrasts; p>0.05). In contrast, the nucleardistribution pattern did not exhibit significant (ANOVA: p>0.05)differences between the groups. However, the perinuclear distributionpattern evidenced significant (ANOVA: p<0.01) differences between thegroups and Tukey-Kramer post host testing revealed differences betweenthe control and noise groups (p<0.01), control and CAE groups (p<0.05),control and CAE+noise groups (p<0.01), noise and CAE group (p<0.01) andnoise and CAE+noise groups (p<0.05). The combined results for CSAsuggest that the number of cells immunopositive for CSA is affected bythe treatment conditions and subcellular distribution.

Discussion

Under normal conditions neurons exhibit high intrinsic metabolicactivity which necessitates intracellular reservoirs of DNA repairproteins [13]. In the current study spiral ganglion neurons wereobserved to compartmentalize DNA repair proteins in their nucleus,cytoplasm and at perinuclear loci. These three intracellularcompartments were embodied by four intracellular distribution patterns.The four patterns were distinct and could be described as nuclear,cytoplasmic, diffuse (both nuclear and cytoplasmic) and perinuclear. Therelevance of these patterns to individual spiral ganglion neurons is notknown. However, each pattern has been reported previously within avariety of human and animal cell types. The nuclear pattern ischaracteristic of some neurons in the cerebral cortex, striatum,hippocampus and cerebellum [12]. It is believed that this pattern mayincrease DNA repair efficiency since the proteins are already localizedin the nucleus [41],[9]. The cytoplasmic pattern is characteristic ofsome human neurons in the dentate gyms and region CA4 of the hippocampusas well as several types of human cell lines [41],[42]. It is believedthat cytoplasmic compartmentalization serves as a reservoir for thetranslocation of repair proteins to the nucleus when needed [14],[43].Additionally, cytoplasmic repair proteins provide protection fornucleotide pools in the cytoplasm as well as mitochondrial DNA (mtDNA)[44]. This is important because cytoplasmic nucleotide pools (e.g.,2′-deoxyribonucleoside-5′-triphospates) are precursors to nuclear DNA(nDNA) and in the cytoplasm these precursors are particularly vulnerableto damage [45],[46]. The diffuse pattern is characteristic of someneurons in the substantia nigra, motor neurons of hypoglossal nucleusand neurons in the ventral tegmental area [12]. The benefit of thisdiffusion pattern is unresolved but it may allow for simultaneousprotection of the nucleus and the cytoplasm. The perinuclear pattern hasbeen demonstrated in human and animal fibroblast cells [47],[14]. Recentexperiments have indicated that perinuclear localization reflectsbinding to mtDNA within mitochondria localized around the nucleus [7].However, there is some evidence that perinuclear localization mayreflect protein binding to the nuclear envelope with residual binding tothe plasma membrane [41],[42]. Nevertheless, perinuclear repair proteinsare believed to serve as a reservoir for both the nucleus and thecytoplasm.

Intracellular stress gradients are known to drive the spatialdistribution of DNA repair proteins. For instance, nuclear localizationindicates more localized stress in the nucleus and cytoplasmiclocalization indicates more localized stress in the cytoplasm [14],[48].This phenomenon seems to be conserved because similar observations havebeen reported for Saccharomyces cerevisiae (yeast) under experimentallyinduced oxidative conditions [17],[10]. For instance when the nucleus ofyeast cells is challenged by reactive oxygen species, DNA repairproteins from the cytoplasm translocate to the nucleus to protect nDNA.Conversely, when the cytoplasm is challenged by oxidative stress, DNArepair proteins in the nucleus translocate to the cytoplasm to defendcytoplasmic pools of DNA. These observations imply that the spatialremodeling of DNA repair proteins is dependent on oxidative demands onthe cell.

It is known that noise exposure increases oxidative demands on spiralganglion neurons [49]. In the current study noise exposure remodeled theendogenous compartmentalization of DNA repair proteins. For instance,under normal conditions the proteins were distributed such that eachintracellular compartment was enriched with at least one repair protein.However, after noise exposure all three proteins were enriched in thecytoplasmic compartment. The relevance of this noise induced effect isnot clear but is associated with neuronal threshold shifts of around 31dB. Treatment with CAE resulted in the enrichment of all three proteinsin multiple intracellular compartments. Interestingly, this specificeffect was associated with significant preservation of neuralsensitivity following noise exposure.

In summary, the current study revealed for the first time that spiralganglion neurons exhibit multiple compartmentalizing modes for DNArepair proteins and these modes were remodeled following noise stressand after DNA repair adjuvant therapy with CAEs. These findings areimportant because DNA repair proteins are necessary for protectingactive genes and preserving cellular functions. Therefore, pharmacologicregulation of the intracellular localization of DNA repair proteins mayrepresent a novel approach to preserving neural function followingstress.

REFERENCES FOR EXAMPLE 2

-   1. Wood R D, Mitchell M, Lindahl T. Human DNA repair genes, 2005.    Mutat Res. 2005; 577:275-283.-   2. Fernandez-Capetillo O, Murga M. Why cells respond differently to    DNA damage: a chromatin perspective. Cell Cycle. 2008; 7:980-983.-   3. Köberle B, Roginskaya V, Wood R D. XPA protein as a limiting    factor for nucleotide excision repair and UV sensitivity in human    cells. DNA Repair 2006; 5:641-648.-   4. Köberle B, Roginskaya V, Zima K S, Masters J R, Wood R D.    Elevation of XPA protein level in testis tumor cells without    increasing resistance to cisplatin or UV radiation. Mol Carcinog.    2008; 47:580-586.-   5. Kang T H, Reardon J T, Sancar A. Regulation of nucleotide    excision repair activity by transcriptional and post-transcriptional    control of the XPA protein. Nucleic Acids Res.-   6. Guthrie O W, Li-Korotky H S, Durrant J D, Balaban C (2008)    Cisplatin induces cytoplasmic to nuclear translocation of nucleotide    excision repair factors among spiral ganglion neurons. Hear Res    239:79-91.-   7. Mirbahai L, Kershaw R M, Green R M, Hayden R E, Meldrum R A,    Hodges N J. Use of a molecular beacon to track the activity of base    excision repair protein OGG1 in live cells. DNA Repair. 2010;    9:144-152.-   8. Tell G, Crivellato E, Pines A, Paron I, Pucillo C, Manzini G,    Bandiera A, Kelley M R, Di Loreto C, Damante G. Mitochondrial    localization of APE/Ref-1 in thyroid cells. Mutat Res. 2001;    485:143-152.-   9. Ahmad A, Enzlin J H, Bhagwat N R, Wijgers N, Raams A, Appledoorn    E, Theil A F, J Hoeijmakers J H, Vermeulen W, J Jaspers N G, Schärer    O D, Niedernhofer L J. Mislocalization of XPF-ERCC1 nuclease    contributes to reduced DNA repair in XP-F patients. PLoS Genet.    2010; 6:e1000871.-   10. Griffiths L M, Swartzlander D, Meadows K L, Wilkinson K D,    Corbett A H, Doetsch P W. Dynamic compartmentalization of base    excision repair proteins in response to nuclear and mitochondrial    oxidative stress. Mol Cell Biol. 2009; 29:794-807.-   11. Guthrie O W. Dys-synchronous regulation of XPC and XPA in    trigeminal ganglion neurons following cisplatin treatment cycles.    Anticancer Res. 2008; 2637-2640.-   12. Yang S Z, Zhang Y F, Zhang L M, Huang Y L, Sun F Y.    Immunohistochemical analysis of nucleotide excision repair factors    XPA and XPB in adult rat brain. Anat Rec (Hoboken). 2008;    291:775-780.-   13. Brooks P J. The case for 8,5′-cyclopurine-2′-deoxynucleosides as    endogenous DNA lesions that cause neurodegeneration in xeroderma    pigmentosum. Neuroscience. 2007; 145:1407-1417.-   14. Seluanov A, Danek J, Hause N, Gorbunova V. Changes in the level    and distribution of Ku proteins during cellular senescence. DNA    Repair. 2007; 6:1740-8.-   15. Swartzlander D B, Griffiths L M, Lee J, Degtyareva N P, Doetsch    P W, Corbett A H. Regulation of base excision repair: Ntgl nuclear    and mitochondrial dynamic localization in response to genotoxic    stress. Nucleic Acids Res. 2010; 38:3963-3974.-   16. Jung Y, Lippard S J. Direct cellular responses to    platinum-induced DNA damage. Chem Rev. 2007; 107:1387-1407.-   17. Swartzlander D B, Griffiths L M, Lee J, Degtyareva N P, Doetsch    P W, Corbett A H. Regulation of base excision repair: Ntgl nuclear    and mitochondrial dynamic localization in response to genotoxic    stress. Nucleic Acids Res. 2010; 38:3963-3974.-   18. Li Z, Musich P R, Zou Y. Differential DNA damage responses in    p53 proficient and deficient cells: cisplatin-induced nuclear import    of XPA is independent of ATR checkpoint in p53-deficient lung cancer    cells. Int J Biochem Mol Biol. 2011; 2:138-145.-   19. Kujawa S G, Liberman M C. Acceleration of age-related hearing    loss by early noise exposure: evidence of a misspent youth. J    Neurosci. 2006; 26:2115-2123.-   20. Kujawa S G, Liberman M C. Adding insult to injury: cochlear    nerve degeneration after “temporary” noise-induced hearing loss. J    Neurosci. 2009; 29:14077-14085.-   21. Akesson, C, Lindgren H, Pero R W, Leanderson T, Ivars F. An    extract of Uncaria tomentosa inhibiting cell division and NF-kappa B    activity without inducing cell death. Int. Immunopharmacol. 2003a;    3: 1889-1900.-   22. Akesson C, Pero R W, Ivars F. C-Med 100, a hot water extract of    Uncaria tomentosa, prolongs lymphocyte survival in vivo.    Phytomedicine. 2003b; 10: 23-33.-   23. Mammone T, Akesson C, Gan D, Giampapa V, Pero R W. A water    soluble extract from Uncaria tomentosa (Cat's Claw) is a potent    enhancer of DNA repair in primary organ cultures of human skin.    Phytother Res. 2006; 20: 178-183.-   24. Pero R W, Giampapa V, Vojdani A. Comparison of a broad spectrum    anti-aging nutritional supplement with and without the action of a    DNA repair enhancing cat's claw extract. J Anti Aging Med. 2002; 5:    345-355.-   25. Pero R W, Amiri A, Sheng Y, Welther M, Rich M. Formulation and    in vitro/in vivo evaluation of combining DNA repair and immune    enhancing nutritional supplements. Phytomedicine. 2005; 12: 255-263.-   26. Pero R W, Lund H, Leanderson T. Antioxidant metabolism induced    by quinic acid. Increased urinary excretion of tryptophan and    nicotinamide. Phytother Res. 2009; 23: 335-346.-   27. Sheng Y, Bryngelsson C, Pero R W. Enhanced DNA repair, immune    function and reduced toxicity of C-MED-100, a novel aqueous extract    from Uncaria tomentosa. J Ethnopharmacol. 2000a; 69: 115-126.-   28. Sheng Y, Pero R W, Wagner H. Treatment of chemotherapy-induced    leukopenia in a rat model with aqueous extract from Uncaria    tomentosa. Phytomedicine. 2000b; 7: 137-143.-   29. Sheng Y, Li L, Holmgren K, Pero R W. DNA repair enhancement of    aqueous extracts of Uncaria tomentosa in a human volunteer study.    Phytomedicine. 2001; 8: 275-282.-   30. Sheng Y, Akesson C, Holmgren K, Bryngelsson C, Giamapa V, Pero    R W. An active ingredient of Cat's Claw water extracts    identification and efficacy of quinic acid. J Ethnopharmacol. 2005;    96: 577-584.-   31. Costa R M, Chiganças V, Galhardo Rda S, Carvalho H, Menck C F.    The eukaryotic nucleotide excision repair pathway. Biochimie 2003;    85:1083-1099.-   32. Hanawalt P C, Spivak G. Transcription-coupled DNA repair: two    decades of progress and surprises. Nat Rev Mol Cell Biol 2008;    9:958-970.-   33. Guthrie O W, Gearhart C A, Fulton S, Fechter L D. Carboxy alkyl    esters of Uncaria tomentosa augment recovery of sensorineural    functions following noise injury. Brain Res. 2011; 1407:97-106.-   34. Chen G D, Fechter L D. The relationship between noise-induced    hearing loss and hair cell loss in rats. Hear Res. 2003; 177: 81-90.-   35. Lorito G, Giordano P, Prosser S, Martini A, Hatzopoulos S.    Noise-induced hearing loss: a study on the pharmacological    protection in the Sprague Dawley rat with N-acetyl cysteine. Acta    Otorhinolaryngol Ital. 2006; 26, 133-139.-   36. Guthrie O W, Carrero-Martínez F A. Real-time quantification of    Xeroderma pigmentosum mRNA from the mammalian cochlea. Ear Hear.    2010; 31:714-721.-   37. Mirkin N, Fonseca D, Mohammed S, Cevher M A, Manley J L, Kleiman    F E. The 3′ processing factor CstF functions in the DNA repair    response. Nucleic Acids Res 2008; 36:1792-1804-   38. Gospodinov A, Ivanov R, Anachkova B, Russev G. Nucleotide    excision repair rates in rat tissues. Eur J Biochem. 2003;    270:1000-1005.-   39. Kasahara T, Kuwayama C, Hashiba M, Harada T, Kakinuma C,    Miyauchi M, Degawa M. The gene expression of hepatic proteins    responsible for DNA repair and cell proliferation in    tamoxifen-induced hepatocarcinogenesis. Cancer Sci. 2003;    94:582-588.-   40. Guthrie O W. DNA repair proteins and telomerase reverse    transcriptase in the cochlear lateral wall of cisplatin-treated    rats. J Chemother. 2009; 21:74-79.-   41. Youssoufian H. Localization of Fanconi anemia C protein to the    cytoplasm of mammalian cells. Proc Natl Acad Sci USA. 1994;    91:7975-7979.-   42. Duguid J R, Eble J N, Wilson T M, Kelley M R. Differential    cellular and subcellular expression of the human multifunctional    apurinic/apyrimidinic endonuclease (APE/ref-1) DNA repair enzyme.    Cancer Res. 1995; 55:6097-6102.-   43. Knudsen N Ø, Andersen S D, Lützen A, Nielsen F C, Rasmussen L J.    Nuclear translocation contributes to regulation of DNA excision    repair activities. DNA Repair. 2009; 8:682-689.-   44. Rai P. Oxidation in the nucleotide pool, the DNA damage response    and cellular senescence: Defective bricks build a defective house.    Mutat Res. 2010; 703:71-81.-   45. Hudson E K, Hogue B A, Souza-Pinto N C, Croteau D L, Anson R M,    Bohr V A, Hansford R G. Age-associated change in mitochondrial DNA    damage. Free Radic Res. 1998; 29:573-579.-   46. Santos J H, Mandavilli B S. Measuring oxidative mtDNA damage and    repair using quantitative PCR. Van Houten B. Methods Mol Biol. 2002;    197:159-176-   47. Cool B L, Sirover M A. Immunocytochemical localization of the    base excision repair enzyme uracil DNA glycosylase in quiescent and    proliferating normal human cells. Cancer Res. 1989; 49:3029-3036.-   48. Frossi B, Tell G, Spessotto P, Colombatti A, Vitale G,    Pucillo C. H(2)O(2) induces translocation of APE/Ref-1 to    mitochondria in the Raji B-cell line. J Cell Physiol. 2002;    193:180-186.-   49. Xiong M, He Q, Lai H, Wang J. Oxidative stress in spiral    ganglion cells of pigmented and albino guinea pigs exposed to    impulse noise. Acta Otolaryngol. 2011; 131:914-920.

Example 3 Materials and Methods

Animals and Experimental Design

Experiments were conducted on male Long-Evans rats (250-300 g) that wereacquired from Harlan Laboratories, Inc. (Livermore, Calif. USA). Allexperimental protocols were approved by the Institutional Animal Careand Use Committee at the Loma Linda Veteran's Affairs Hospital. A totalof 48 animals were used in the experiments. After arriving at thevivarium, the animals were allowed to acclimate for one week. Baselinedistortion product otoacoustic emissions (DPOAE) were then collected oneach animal to verify cochlear function. The animals were assigned toone of four groups based on their DPOAE recordings in order tocounterbalance cochlear function between the groups. The four groupsincluded a CAE group (n=10), a noise+CAE group (n=11), a control group(n=14) and a noise exposure group (n=13). A formulation of CAE that hasbeen standardized to increase DNA repair activity was prepared byOptigenex Inc. (Hoboken, N.J. USA) as reported previously (Guthrie etal., 2011; Sheng et al., 2005). The animals in the CAE and CAE+noisegroups were administered 160 mg/kg of CAE by gastric gavage for 28consecutive days (Guthrie et al., 2011; Sheng et al, 2000a). The controlgroup was also treated via gastric gavage with distilled water(dissolving agent for CAE) for 28 consecutive days. The noise group didnot receive any treatment beyond being exposed to the nose dose (seebelow). Noise exposure of the noise and CAE+noise groups occurred on the29th day (one day after the 28 days of water or CAE treatment). Thenpost-exposure DPOAE measurements and tissue harvesting were conductedfrom each group on the 30th day (1-day after the noise exposure).Tissues were harvested to evaluate whether or not γ-H2Ax expressionincreased in the organ of Corti after noise exposure and whether or notCAE treatment could reduce the expression of γ-H2Ax in the organ ofCorti at an early time point (1-day post trauma) after the exposure. Toevaluate whether a putative effect on γ-H2Ax expression is associatedwith long-term recovery of cochlear function, some of the animals wereallowed to survive for 4 weeks post-noise exposure and cochlear functionwas evaluated again with DPOAE. Table 3 describes the different animalgroups, their treatment regimen and the experimental design.

TABLE 3 Experimental design. DPOAE CAE recovery Baseline treatment NoiseDPOAE + 1-week and DPOAE 28 days of Day Sacrifice 4-week Groups Day 0treatment 29 Day 30 post-noise Control (N = 14) water + (N = 9) Noise (N= 13) noise + (N = 8) CAE (N = 10) CAE + Noise + CAE (N = 11) CAEnoise + (N = 6) Abbreviations: CAE, Carboxy alkyl esters; DPOAE,distortion product otoacoustic emissions.

Distortion Product Otoacoustic Emissions (DPOAE)

The cubic 2ƒ₁-ƒ₂ DPOAE is particularly sensitive to cochlear noisedamage. Therefore DPOAEs (2ƒ₁-ƒ₂ level as a function of increasingstimulus frequency, commonly referred to as a DP-gram) were recorded asdescribed previously (Guthrie and Xu, 2012). Briefly, each animal wasanesthetized with ketamine/xylazine (44/7 mg/kg, im.) while normal bodytemperature was maintained using a direct current (dc) heating unitbuilt into the surgical table. The cubic 2ƒ₁-ƒ₂ DPOAE was recorded withtwo primaries, ƒ₂ and ƒ₁; where ƒ₂ is higher than ƒ₁ at an ƒ₂/ƒ₁ ratioof 1.25. The primaries (L) were presented in 0.1-octave increments from3.2 to 63 kHz. The levels of the primaries were set to L₁−10=L₂ asindicated in the figures. The frequency and level ratios of theprimaries were selected to maximize the 2ƒ₁-ƒ₂ SPL recorded from the earcanal (Whatehead et al., 1995a, 1995b, and 1995c). A customized signalpresentation, acquisition and analysis program written in LabVIEWversion 7.1 (National Instruments, Austin, Tex. USA) was used to drive aPCI-4461 computer-based DSP board (National Instruments, Austin, Tex.,USA) for generation of the primaries and Fourier analysis of theresponse.

Noise Exposure

Both the noise-only group and the CAE+noise group were exposed in thesame noise exposure chamber at the same time. The noise exposureparadigm has been described previously (Guthrie et al., 2011). Briefly,the animals were exposed to an octave band of noise (OBN) centered at 8kHz. The intensity of the noise was 105 dB SPL and the duration was 4hours. The animals were awake during the exposure and were free to movearound in a wire-cloth enclosure within a 40 L noise chamber. The noisewas generated by a Function Generator (Sanford Research System, MenloPark, Calif. USA) coupled to a Frequency Device (Frequency Device Inc.,Haverhill, Mass. USA). Vifa D25AG-05 speakers (Vifa International A/S,Videbaek, Denmark) located approximately 5 cm above the animals'wire-cloth enclosure was used to present the noise. The frequencyspectrum of the noise was verified in the noise chamber containing therats with a sound level meter (Quest Electronics, Oconomowoc, Wis. USA)close to the animals' pinnae.

Immunolabeling

Animal and Tissue Preparation

Immunolabeling of γ-H2Ax within cells in tissue sections is a standardmethod of detecting DSB (Wang et al., 2009; Redon et al., 2011; Redon etal., 2012). Therefore, we employed γ-H2Ax immunolabeling andquantification to determine differences between the groups. Twentyanesthetized animals (control group=5, noise group=5, CAE group=5 andCAE+noise group=5) were sacrificed by transcardial perfusion withphosphate-buffered saline (PBS; 10 mM, pH 7.4) followed byperiodate-lysine-paraformaldehyde fixative (Guthrie, 2008a). The headswere then removed, skinned and post-fixed in 4% paraformaldehydeovernight at 22° C. Formic acid (10%) was used for chemicaldecalcification of the heads as described previously (Guthrie and Xu,2012). Then the heads were paraffin embedded and sectioned at 5 μm inthe midmodiolar plane. The sections were incubated in a heated waterbath and mounted on subbed slides for immunolabeling. Kidney tissueswere simultaneously harvested, postfixed, paraffin embedded, sectionedand mounted on subbed slides.

Immunoperoxidase Procedure

Tissue sections were de-paraffinized in zylene, hydrated in graded ethylalcohol and water then incubated in 0.9% H₂O₂ for 10 minutes. They werethen heated for 20 minutes at 90-98° C. in a low pH (0.80-3.06) sodiumcitrate-citric acid buffer (antigen retrieval) and then rinsedthoroughly with PBS. Afterwards, the sections were pre-treated with ablocking solution of normal goat serum, 10% Triton X-100 and 2% bovineserum albumin (BSA; Sigma, St. Louis, Mo., USA) in PBS for 1 hour. Theprimary antibody was diluted in the blocking solution at a 1:100concentration. The primary antibody (anti-γ-H2Ax, Ser139) iscommercially available (Santa Cruz Biotechnology, Inc., Santa Cruz,Calif. USA). The specificity of the antibody has been confirmedpreviously (Verma et al., 2010; Li et al., 2011; Gupta, et al., 2011).Nevertheless, negative control experiments were conducted such thatsections were incubated with blocking serum without the primary antibody(Guthrie et al., 2008). Such negative control sections were processed atthe same time as the experimental sections and received simultaneous andidentical treatments. Furthermore, positive control experiments wereconducted by immunolabeling tissue that is known to exhibit constitutiveexpression of γ-H2Ax (Dmitrieva et al., 2004; see FIG. 12). Afterincubating the sections with the primary antibody (or blocking serum fornegative control sections) for 48 hours at 4° C., the sections wererinsed with PBS. They were then treated with a biotinylated anti-rabbitsecondary antibody (Vector Laboratories, Temecula, Calif. USA) diluted1:100 in PBS+2% BSA for 24 hours at 4° C. The sections were then rinsedin PBS, incubated with preformed avidin-biotin-peroxidase enzymecomplexes (Vectastain ABC reagent; Vector Laboratories, Inc.,Burlingame, Calif. USA) for one hour, rinsed again with PBS and thentreated with a solution of Trizma pre-set crystals (1.58 g;Sigma-Aldrich, St. Louis, Mo. USA) to stabilize peroxidase enzymereactions. The peroxidase enzyme complexes were then used to oxidize3,3′-diaminobenzidine tetrahydrochloride to produce a brown chromogen.

Quantitative Morphometry

Equipment.

A Leica DM2500 upright microscope (Leica Microsystems Inc., Bannockburn,Ill. USA) was used for brightfield microscopy. A ProgRes® CF^(scan)digital camera (JENOPTIK Laser, Jena, Germany) mounted on the LeicaDM2500 was used for digital image capturing. Image-Pro® plus version 6.3(Media Cybernetics Inc., Bethesda, Md., USA) for Windows™ was used tocontrol image capturing, pixel thresholding and bitmap analyses. A DellOptiplex GX620 with an Intel Core2 processor was used for softwareoperations.

Photomicroscopy.

The microscope current drain, light source and temperature werestandardized to ensure accurate and consistent reading of each tissuesection. The intensity of the light to a 1.4 megapixelcharged-coupled-device color sensor was monitored over an 8 hour periodby capturing a blank field (cover slip, mounting media and glass slide).The light intensity fluctuated by only 0.1% (a modest difference in meangray value of 0.2748) within the first hour of being turned on thenstabilized when the lamp housing reached a temperature of 62±1° C. Allphotomicrographs were taken when the microscope light intensitystabilized. To further ensure consistent spatial and temporalillumination for each tissue section during microscopy, the mean grayvalue (g) for blank fields adjacent to each tissue section wasmaintained at ≧254 g. In addition to providing consistent illumination,this high level illumination had the added advantage of maskingbackground staining which was significantly lighter (light brown stain)than the antibody staining which exhibited a heavy brown stain and thusunmasked by the illumination. Photomicrographs of the organ of Cortiwere taken with an N-PLAN 40×/0.65 objective lens. Thesephotomicrographs (680×512 pixels) were saved in uncompressedtagged-image file format for later retrieval. They were then convertedfrom 48 bits/pixel to 24 bits for subsequent analyses. To further removebackground staining from each photomicrograph a predetermined thresholdcriterion was applied via Image-Pro's threshold algorithm. Thisthreshold criterion was empirically determined from pilot experiments.For instance, the acellular tectorial membrane of Corti's organ issometimes stained after the immunolabeling procedure. This is due to thetrapping of the secondary antibody within the microfibrillar matrix ofthe tectorial membrane and the failure of wash steps to completelydislodge the secondary antibody. The thresholding criteria employed inthis study was trained on masking background levels of staining such asthe level of background found in the tectorial membrane. Therefore,applying the threshold criteria increased the signal-to-noise ratio.After thresholding, an area of interest (AOI) field was selected withineach organ of Corti. Bitmap readings were then taken within the AOI. Thebitmaps record the positional matrix (n₁×n₁) and brightness (b) of eachprimary colored (red, green and blue or RGB) pixel (n₁×n₁×3). Thesebitmap readings were then used in the determination of absolutechromogen levels by the cumulative signal strength technique.

Cumulative Signal Strength.

Cumulative signal strength is an objective technique that quantifies theabsolute amount of immunolabeling (staining intensity) inphotomicrographs (Matkowskyj et al., 2000; Matkowskyj et al., 2003).This is achieved by measuring the mathematical energy (E) within eachn₁×n₁×3 pixel of an N₁×N₁×3 photomicrograph. The computation is asfollows (Hu et al., 2008);

$\begin{matrix}{E = {\sqrt{\frac{1}{N_{1}N_{2}}{\sum\limits_{n_{1} = 1}^{N_{1}}{\sum\limits_{n_{2} = 1}^{N_{2}}\left\lbrack {I_{R}\left( {n_{1},n_{2}} \right)} \right\rbrack^{2}}}} + \sqrt{\frac{1}{N_{1}N_{2}}{\sum\limits_{n_{1} = 1}^{N_{1}}{\sum\limits_{n_{2} = 1}^{N_{2}}\left\lbrack {I_{G}\left( {n_{1},n_{2}} \right)} \right\rbrack^{2}}}} + \sqrt{\frac{1}{N_{1}N_{2}}{\sum\limits_{n_{1} = 1}^{N_{1}}{\sum\limits_{n_{2} = 1}^{N_{2}}\left\lbrack {I_{B}\left( {n_{1},n_{2}} \right)} \right\rbrack^{2}}}}}} & (1)\end{matrix}$where I_(i)(n₁,n₂) is the energy from a particular hue (i=R, G or B) atposition n₁×n₂ in the AOI frame. In photomicrographs from bright fieldmicroscopy I_(i)(n_(i),n₂)=255−b_(i)(n₁,n₂), where b is the brightnessvalue or gray value. The E that reflects specific staining of the γ-H2Axantibody (E_(γ-H2Ax)) is the absolute value of the difference betweenantibody induced immunolabeling (E_(antibody[+])) and that induced bythe absence of the antibody (E_(antibody[−])) also known as the negativecontrol;E _(γ-H2Ax)=(E _(antibody[+]))−(E _(antibody[−]))  (2)

Statistical Analysis

Statistical analyses were conducted with Prism 5, version 5.03 (GraphPadSoftware, Inc., La Jolla, Calif. USA). The DPOAE data was analyzed forgroup effects such that analysis of variance (ANOVA) testing wasconducted on 2ƒ₁-ƒ₂ DPOAE levels to determine significant differencesbetween the groups. Post-hoc testing employed Dunnett's pairedcomparison analyses. The intensity of γ-H2Ax immunolabeling was recordedfrom midmodiolar cochlear sections. For each animal, duplicate readingswere recorded and 10 cochlear sections were used for each group (a totalof 40 sections). The apical, middle and basal cochlear coils wereevident in the sections therefore, 120 (40×3) cochlear coils werestudied. Statistical differences between groups were determined withANOVA and Bonferroni's multiple comparison testing.

Results

γ-H2Ax Labeling

It is known that the kidney exhibit consistent immunolabeling of γ-H2Axwhich is believed to represent DSB that form in noncoding regions of thegenome called gene deserts (Dmitrieva et al., 2011; Dmitrieva et al.,2004). Therefore, kidney sections (FIG. 12) served as positive andnegative controls for the immunolabeling experiments. FIG. 12A revealsγ-H2Ax labeling in podocytes of the renal corpulse (positive control).This labeling was abolished when the antibody is omitted from theimmunolabeling procedure (FIG. 12B, negative control). Immunolabelingfor γ-H2Ax was also detected within the organ of Corti. The labeling waspresent under normal (control) conditions and after noise or CAEtreatment. FIG. 13A is a representative example of the staining Severalcochlear structures are labeled but labeling in the organ of Corti isparticularly prominent. Panels B and C from FIG. 13 demonstrates thatγ-H2Ax labeling could be detected within hair cells and supporting cellswith and without noise exposure. This persistent expression of γ-H2Ax isconsistent with previous research that has shown persistent expressionof DNA repair proteins in the organ of Corti (Guthrie, 2008b; Guthrie,2009; Guthrie and Carrero-Martinez, 2010).

γ-H2Ax Levels

The prominence of γ-H2Ax labeling in the organ of Corti made itdifficult to make subjective judgments on the level of expressionbetween the groups. Therefore, to determine differences in stainingintensity, the level of γ-H2Ax in the organ of Corti was quantified bydetermining the absolute amount of chromogen per pixel by employing thecumulative signal strength technique (Matkowshyj et al., 2000;Matkowskyj et al., 2003; Hu et al., 2008). This technique measures thesignal energy as a function of pixel, where energy is defined in themathematical sense. Therefore, the values are unit less and thusreported as mathematical energy units per pixel (E_(M)/pix). FIG. 14Areveals that γ-H2Ax level in the control group was similar to that inthe CAE group. However, noise exposure induced an increase in γ-H2Ax.This increase was also observed after co-treatment with CAE and noise.Note that γ-H2Ax level was lower in the CAE+noise treated group relativeto that in the noise-only group. FIG. 14B reveals γ-H2Ax levels for eachcochlear turn (apical, middle and basal coils) as a function of thetreatment groups. In all cochlear turns, γ-H2Ax levels were similarbetween the control and CAE groups. However, γ-H2Ax levels were highestamong the noise and CAE+noise groups. In the middle and basal turnγ-H2Ax levels were higher in the noise group compared to the CAE+noisegroup. Therefore, inspection of individual cochlear turns furthersupported the notion that CAE treatment reduced noise induced levels ofγ-H2Ax.

Statistical analyses were conducted on γ-H2Ax level measurements. Aone-way ANOVA where treatment condition served as a between subjectsfactor revealed that there was a statistically significant difference inγ-H2Ax levels between the four groups (F_([3,36])=30.33, p<0.01).Bonferroni pairwise contrast revealed no statistically significantdifferences between the control and CAE groups (p>0.05). But thesegroups exhibited significantly lower γ-H2Ax levels compared to the noiseexposed group (p<0.01). Furthermore, the noise exposed group exhibitedsignificantly higher γ-H2Ax levels compared to the CAE+noise group(p<0.05). These statistical calculations suggest that CAE treatmentreduced noise induced γ-H2Ax within the organ of Corti. This conclusionwas further supported by statistical calculations on γ-H2Ax levelmeasurements from individual cochlear turns. For instance, the controland CAE group showed no significant difference (p>0.05) in γ-H2Ax levelsregardless of cochlear turn (apical, middle or basal). However, thenoise group and the CAE+noise group were significantly (p<0.05)different at the basal and middle turns. In these cochlear turns, γ-H2Axwas significantly (p<0.05) lower within the CAE+noise group. The pooledresults indicate that CAE treatment reduced noise induced γ-H2Ax levelsin the basal and middle cochlear turns. This is significant becausebasalward turns are known to be more vulnerable to cell death and theloss of cochlear function after exposure to the same noise dose used inthe current study (Guthrie et al., 2011).

Protection From Noise Injury

To evaluate protection from noise injury, DP-grams of 2ƒ₁-ƒ2 wererecorded. These recordings were obtained at baseline and 1 day followingnoise exposure. FIG. 15 shows the response of 2ƒ₁-ƒ₂ DPOAE as a functionof ƒ₂ frequency driven with primary levels (L) at 65/55 dB SPL. Baselinerecordings revealed that all groups had large 2ƒ₁-ƒ₂ levels thatexceeded the noise floor by at least 6 dB. At 1 day post-noise exposure,the control groups (vehicle-control and CAE treated groups) showedequivalent 2ƒ₁-ƒ2 levels (two-way ANOVA: F_([1,43])=2.09, p>0.05).However there was a significant difference (two-way ANOVA:F_([1,43])=56.37, p<0.01) between the noise treated groups where theCAE+noise group exhibited higher (better) levels than the noise-onlygroup. Furthermore, the highest frequency components (≧32 kHz) of thenoise-only group was suppressed into the noise floor (average=1.27 dBabove noise floor) while that of the CAE+noise group remained above thenoise floor (average=7.31 dB above noise floor). These findings suggestthat 2ƒ₁-ƒ₂ DPOAE levels from the CAE+noise group was better preservedthan that of the noise group as early as 1 day post-noise exposure.

Recovery From Noise Injury

Recordings of the 2ƒ₁-ƒ₂ DPOAE were conducted out to 4 weeks post-noiseexposure to evaluate functional recovery from the noise exposure. Thestimulus primary levels were set at 55/35 (L₁/L₂) which increases thesensitivity of the 2ƒ₁-ƒ₂ recordings (Avan et al., 2003). FIG. 16illustrates the change in 2ƒ₁-ƒ₂ levels for three time points (1-day,1-week and 4-weeks post noise exposure). In the control group, 2ƒ₁-ƒ2levels were robust and exhibited modest variations between time points.However, in the noise exposed group there was wide-spread loss of 2ƒ₁-ƒ₂levels at 1 day post exposure. For instance, the 2ƒ₁-ƒ₂ DPOAE levelswere reduced to approximate the noise floor over the entire ƒ₂ frequencyrange. At 1 week post-noise exposure there was some recovery in 2ƒ₁-ƒ₂levels but the frequency range between ˜8-24 kHz showed prominent loss.At 4 weeks post-noise exposure there was still a prominent loss in the˜8-24 kHz range. These results indicate that the noise exposure resultedin permanent loss 1½-octave above the center frequency (8 kHz) of theOBN. Indeed, permanent cochlear loss in Long-Evans rats exposed to an 8kHz OBN at 105 dB SPL for 4 hours typically occurs at 4 weekspost-exposure (Chen and Fechter, 2003; Lorito et al., 2006).

The capacity of the CAE+noise group to recover from noise injury isshown in FIG. 16. At 1 day post-noise exposure the CAE+noise groupshowed loss of 2ƒ₁-ƒ₂ levels. For instance, frequencies below ˜16 kHzwere suppressed in to the noise floor. However, high frequencies between˜16-32 kHz were not reduced into the noise floor which is in contrastwith the noise-only treatment where the entire frequency spectrum wassuppressed into the noise floor. These data support that of FIG. 15 insuggesting that CAE may provide early protection against noise injury.Indeed, at only 1 week post-noise exposure the CAE+noise group showedrecovery while the noise-only group still exhibited a prominent loss.One week following noise exposure is considered an early time pointbecause functional recovery is still occurring. Interestingly, theCAE+noise group showed almost complete recovery of 2ƒ₁-ƒ₂ levels at 4weeks post-noise exposure. This indicates that the CAE treated group wasable to recover from the noise exposure.

Statistical analyses were conducted on the 2ƒ₁-ƒ₂ levels obtained at 4weeks post noise exposure. A one-way ANOVA revealed significant(F_([2,129])=11.17, p<0.01) main effects for the groups. Dunnett'smultiple comparison testing demonstrated that there was a statisticallysignificant (p<0.01) difference between the control and noise groups butthere was no significant (p>0.05) difference between the control andCAE+noise groups.

REFERENCES FOR EXAMPLE 3

-   Akesson, C., Lindgren, H., Pero, R. W., Leanderson, T., Ivars, F.,    2003a. An extract of Uncaria tomentosa inhibiting cell division and    NF-kappa B activity without inducing cell death. Int.    Immunopharmacol. 3, 1889-900.-   Akesson C, Pero R W, Ivars F., 2003b. C-Med 100, a hot water extract    of Uncaria tomentosa, prolongs lymphocyte survival in vivo.    Phytomedicine. 10: 23-33.-   Avan P, Bonfils P, Gilain L, Mom T. 2003. Physiopathological    significance of distortion-product otoacoustic emissions at 2f1-f2    produced by high-versus low-level stimuli. J Acoust Soc Am. 113:    430-441.-   Chen G D, Fechter L D., 2003. The relationship between noise-induced    hearing loss and hair cell loss in rats. Hear Res. 177: 81-90.-   Crowe, S. L., Movsesyan, V. A., Jorgensen, T. J., Kondratyev,    A., 2006. Rapid phosphorylation of histone H2A.X following    ionotropic glutamate receptor activation. Eur J Neurosci 23,    2351-1361.-   Dmitrieva N I, Cai Q, Burg M B. 2004. Cells adapted to high NaCl    have many DNA breaks and impaired DNA repair both in cell culture    and in vivo. Proc Natl Acad Sci USA. 101: 2317-2322.-   Dmitrieva N I, Cui K, Kitchaev D A, Zhao K, Burg M B., 2011. DNA    double-strand breaks induced by high NaCl occur predominantly in    gene deserts. Proc Natl Acad Sci USA. 108:20796-20801.-   Firsanov D, Vasilishina A, Kropotov A, Mikhailov V., 2012. Dynamics    of γH2AX formation and elimination in mammalian cells after    X-irradiation. Biochimie. 94: 2416-2422.-   Frenzilli, G., Lenzi, P., Scarcelli, V., Fornai, F., Pellegrini, A.,    Soldani, P., Paparelli, A., Nigro, M., 2004. Effects of loud noise    exposure on DNA integrity in rat adrenal gland. Environ Health    Perspect, 112:1671.-   Fryatt, A. G., Mulheran, M., Egerton, J., Gunthorpe, M. J.,    Grubb, B. D., 2011. Ototrauma induces sodium channel plasticity in    auditory afferent neurons. Mol Cell Neurosci 48, 51-61.-   Ghabili, K., Shoja, M. M., Tubbs, R. S., Rahimi-Ardabili, B.,    Ansarin, K., 2007. Sonocarcinogenesis: loud noise may cause    malignant transformation of cells. Med Hypotheses 69, 1156.-   Gupta, K., Chakrabarti, A., Rana, S., Ramdeo, R., Roth, B. L.,    Agarwal, M. L., Tse, W., Agarwal, M. K., Wald, D. N., 2011.    Securinine, a myeloid differentiation agent with therapeutic    potential for AML. PLoS One 6, e, 21203.-   Guthrie O W, Xu H., 2012. Noise exposure potentiates the subcellular    distribution of nucleotide excision repair proteins within spiral    ganglion neurons. Hear Res. 294: 21-30.-   Guthrie O W, Gearhart C A, Fulton S, Fechter L D. 2011. Carboxy    alkyl esters of Uncaria tomentosa augment recovery of sensorineural    functions following noise injury. Brain Res. 1407:97-106.-   Guthrie O W, Carrero-Martínez F A., 2010. Real-time quantification    of Xeroderma pigmentosum mRNA from the mammalian cochlea. Ear Hear.    31: 714-721.-   Guthrie O W., 2009. DNA repair proteins and telomerase reverse    transcriptase in the cochlear lateral wall of cisplatin-treated    rats. J Chemother. 21: 74-79.-   Guthrie O W., 2008a. Dys-synchronous regulation of XPC and XPA in    trigeminal ganglion neurons following cisplatin treatment cycles.    Anticancer Res. 2637-2640.-   Guthrie O W., 2008b. Preincision complex-I from the excision    nuclease reaction among cochlear spiral limbus and outer hair cells.    J Mol Histol. 39:617-625.-   Guthrie O W., 2008c. Aminoglycoside induced ototoxicity. Toxicology,    249(2-3):91-96.-   Guthrie O W, Li-Korotky H S, Durrant J D, Balaban C., 2008.    Cisplatin induces cytoplasmic to nuclear translocation of nucleotide    excision repair factors among spiral ganglion neurons. Hear Res.    239, 79-91.-   Han, W., Shi, X., Nuttall, A. L., 2006. AIF and endoG translocation    in noise exposure induced hair cell death. Hear Res 211, 85-95.-   Hatahet, Z., Purmal, A. A., Wallace, S. S., 1994. Oxidative DNA    lesions as blocks to in vitro transcription by phage T7 RNA    polymerase. Ann N Y Acad Sci 726, 346-348.-   Henderson, D., Bielefeld, E. C., Harris, K. C., Hu, B. H., 2006. The    role of oxidative stress in noise-induced hearing loss. Ear Hear 27,    1-19.-   Huang, H., Das, R. S., Basu, A. K., Stone, M. P., 2011. Structure of    (5′S)-8,5′-cyclo-2′-deoxyguanosine in DNA. J Am Chem Soc 133,    20357-20368.-   Hu, B. H., Henderson, D., Nicotera, T. M., 2006. Extremely rapid    induction of outer hair cell apoptosis in the chinchilla cochlea    following exposure to impulse noise. Hear Res 211, 16-25.-   Hu J J, Ambrus A, Fossum T W, Miller M W, Humphrey J D, Wilson    E., 2008. Time courses of growth and remodeling of porcine aortic    media during hypertension: a quantitative immunohistochemical    examination. J Histochem Cytochem. 56: 359-370.-   Kathe S D, Shen G P, Wallace S S., 2004. Single-stranded breaks in    DNA but not oxidative DNA base damages block transcriptional    elongation by RNA polymerase II in HeLa cell nuclear extracts. J    Biol Chem. 279: 18511-18520.-   Kenyon, J., Gerson, S. L., 2007. The role of DNA damage repair in    aging of adult stem cells. Nucleic Acids Res 35, 7557-7565-   Khanna K K, Jackson S P. 2001. DNA double-strand breaks: signaling,    repair and the cancer connection. Nat Genet. 27: 247-254.-   Koike M, Mashino M, Sugasawa J, Koike A., 2008. Histone H2AX    phosphorylation independent of ATM after X-irradiation in mouse    liver and kidney in situ. J Radiat Res. 49: 445-449.-   Lamm, S., Sheng, Y., Pero, R. W., 2001. Persistent response to    pneumococcal vaccine in individuals supplemented with a novel water    soluble extract of Uncaria tomentosa, C-Med-100. Phytomedicine. 8,    267-274.-   Lee, S. C., Bohne, B. A., Harding, G. W., 2008. Cochlear base-apex    differences in cell death pathways following exposure to    low-frequency noise. Otorhinolaryngol J 2, 29-43.-   Le Prell, C. G., Yamashita, D., Minami, S. B., Yamasoba, T.,    Miller, J. M., 2007. Mechanisms of noise-induced hearing loss    indicate multiple methods of prevention. Hear Res 226, 22-43.-   Li C, Fan S, Owonikoko T K, Khuri F R, Sun S Y, Li R. 2011.,    Oncogenic role of EAPII in lung cancer development and its    activation of the MAPK-ERK pathway. Oncogene, 30: 3802-3812.-   Lomonaco, S. L., Xu, X. S., Wang, G., 2009. The role of Bcl-x(L)    protein in nucleotide excision repair-facilitated cell protection    against cisplatin-induced apoptosis. DNA Cell Biol 28, 285-294.-   Lorito G, Giordano P, Prosser S, Martini A, Hatzopoulos S., 2006.    Noise-induced hearing loss: a study on the pharmacological    protection in the Sprague Dawley rat with N-acetyl-cysteine. Acta    Otorhinolaryngol Ital. 26: 133-139.-   Mammone T, Akesson C, Gan D, Giampapa V, Pero R W., 2006. A water    soluble extract from Uncaria tomentosa (Cat's Claw) is a potent    enhancer of DNA repair in primary organ cultures of human skin.    Phytother Res. 20: 178-183.-   Matkowskyj K A, Cox R, Jensen R T, Benya R V., 2003. Quantitative    immunohistochemistry by measuring cumulative signal strength    accurately measures receptor number. J Histochem Cytochem.    51:205-214.-   Matkowskyj K A, Schonfeld D, Benya R V., 2000. Quantitative    immunohistochemistry by measuring cumulative signal strength using    commercially available software photoshop and matlab. J Histochem    Cytochem. 48: 303-312.-   Murai, N., Kirkegaard, M., Järlebark, L., Risling, M., Suneson, A.,    Ulfendahl, M., 2008. Activation of JNK in the inner ear following    impulse noise exposure. J Neurotrauma 25, 72-77.-   Pero, R. W., Amiri, A., Sheng, Y., Welther, M., Rich, M., 2005.    Formulation and in vitro/in vivo evaluation of combining DNA repair    and immune enhancing nutritional supplements. Phytomedicine. 2005,    12, 255-263.-   Pero R W, Giampapa V, Vojdani A., 2002. Comparison of a broad    spectrum anti-aging nutritional supplement with and without the    action of a DNA repair enhancing cat's claw extract. J Anti Aging    Med. 5: 345-355.-   Pero R W, Lund H, Leanderson T., 2009. Antioxidant metabolism    induced by quinic acid. Increased urinary excretion of tryptophan    and nicotinamide. Phytother Res. 23: 335-346.-   Preston-Martin, S., Thomas, D. C., Wright, W. E., Henderson, B.    E., 1989. Noise trauma in the aetiology of acoustic neuromas in men    in Los Angeles County. Br J Cancer 59, 783-786.-   Rogakou E P, Boon C, Redon C, Bonner W M. 1999. Megabase chromatin    domains involved in DNA double-strand breaks in vivo. J Cell Biol.    146: 905-916.-   Rogakou E P, Pilch D R, Orr A H, Ivanova V S, Bonner W M. 1998. DNA    double-stranded breaks induce histone H2AX phosphorylation on    serine 139. J Biol Chem. 273: 5858-5868.-   Rothkamm K, Löbrich M. 2003. Evidence for a lack of DNA    double-strand break repair in human cells exposed to very low x-ray    doses. Proc Natl Acad Sci USA. 100: 5057-5062.-   Redon C E, Nakamura A J, Martin O A, Parekh P R, Weyemi U S, Bonner    W M. 2011. Recent developments in the use of γ-H2AX as a    quantitative DNA double-strand break biomarker. Aging 3: 168-174.-   Redon C E, Weyemi U, Parekh P R, Huang D, Burrell A S, Bonner    W M. 2012. γ-H2AX and other histone post-translational modifications    in the clinic. Biochim Biophys Acta. 1819:743-756.-   Satou K, Hori M, Kawai K, Kasai H, Harashima H, Kamiya H., 2009.    Involvement of specialized DNA polymerases in mutagenesis by    8-hydroxy-dGTP in human cells. DNA Repair (Amst). 8: 637-642.-   Sheng Y, Akesson C, Holmgren K, Bryngelsson C, Giamapa V, Pero R    W., 2005. An active ingredient of Cat's Claw water extracts    identification and efficacy of quinic acid. J Ethnopharmacol. 96:    577-584.-   Sheng Y, Bryngelsson C, Pero R W., 2000a. Enhanced DNA repair,    immune function and reduced toxicity of C-MED-100, a novel aqueous    extract from Uncaria tomentosa. J Ethnopharmacol. 69: 115-126.-   Sheng Y, Li L, Holmgren K, Pero R W, 2001. DNA repair enhancement of    aqueous extracts of Uncaria tomentosa in a human volunteer study.    Phytomedicine, 8: 275-282.-   Sheng Y, Pero R W, Wagner H., 2000b. Treatment of    chemotherapy-induced leukopenia in a rat model with aqueous extract    from Uncaria tomentosa. Phytomedicine, 7: 137-143.-   Stiff T, O'Driscoll M, Rief N, Iwabuchi K, Löbrich M, Jeggo    P A. 2004. ATM and DNA-PK function redundantly to phosphorylate H2AX    after exposure to ionizing radiation. Cancer Res. 64: 2390-2396.-   Taggart, R. T., McFadden, S. L., Ding, D. L., Henderson, D., Jin,    X., Sun, W., Salvi, R., 2001. Gene Expression Changes in Chinchilla    Cochlea from Noise-Induced Temporary Threshold Shift. Noise Health.    3, 1-18.-   Van Campen, L. E., Murphy, W. J., Franks, J. R., Mathias, P. I.,    Toraason, M. A., 2002. Oxidative DNA damage is associated with    intense noise exposure in the rat. Hear Res 164, 29-38.-   Verma R, Rigatti M J, Belinsky G S, Godman C A, Giardina C., 2010.    DNA damage response to the Mdm2 inhibitor nutlin-3. Biochem    Pharmacol. 79, 565-574.-   Wang C, Jurk D, Maddick M, Nelson G, Martin-Ruiz C, von    Zglinicki T. 2009. DNA damage response and cellular senescence in    tissues of aging mice. Aging Cell. 8: 311-323.-   Wang, J., Ruel, J., Ladrech, S., Bonny, C., van de Water, T. R.,    Puel, J. L., 2007. Inhibition of the c-Jun N-terminal    kinase-mediated mitochondrial cell death pathway restores auditory    function in sound-exposed animals. Mol Pharmacol 71, 654-666.-   Whitehead, M. L., Stagner, B. B., Lonsbury-Martin, B. L., Martin, G.    K., 1995a. Effects of ear-canal standing waves on measurements of    distortion-product otoacoustic emissions. J Acoust Soc Am. 98,    3200-3214.-   Whitehead, M. L., Stagner, B. B., McCoy, M. J., Lonsbury-Martin, B.    L., Martin, G. K., 1995b. Dependence of distortion-product    otoacoustic emissions on primary levels in normal and impaired    ears. II. Asymmetry in L1,L2 space. J Acoust Soc Am. 97, 2359-2377.-   Whitehead, M. L., McCoy, M. J., Lonsbury-Martin, B. L., Martin, G.    K., 1995c. Dependence of distortion-product otoacoustic emissions on    primary levels in normal and impaired ears. I. Effects of decreasing    L2 below L1. J Acoust Soc Am. 97, 2346-2358.-   Wang X, Michaelis E K., 2010. Selective neuronal vulnerability to    oxidative stress in the brain. Front Aging Neurosci., 2:12.-   Yamashita, D., Miller, J. M., Jiang, H. Y., Minami, S. B., Schacht,    J., 2004. AIF and EndoG in noise-induced hearing loss. Neuroreport    15, 2719-2722.

All of the compositions and methods disclosed and claimed herein can bemade and executed without undue experimentation in light of the presentdisclosure. While the compositions and methods of this invention havebeen described in terms of preferred embodiments, it will be apparent tothose of skill in the art that variations may be applied to thecompositions and methods and in the steps or in the sequence of steps ofthe method described herein without departing from the spirit and scopeof the invention. More specifically, the described embodiments are to beconsidered in all respects only as illustrative and not restrictive. Allsimilar substitutes and modifications apparent to those skilled in theart are deemed to be within the spirit and scope of the invention asdefined by the appended claims.

All patents, patent applications, and publications mentioned in thespecification are indicative of the levels of those of ordinary skill inthe art to which the invention pertains. All patents, patentapplications, and publications, including those to which priority oranother benefit is claimed, are herein incorporated by reference to thesame extent as if each individual publication was specifically andindividually indicated to be incorporated by reference.

The invention illustratively described herein suitably may be practicedin the absence of any element(s) not specifically disclosed herein.Thus, for example, in each instance herein any of the terms“comprising”, “consisting essentially of”, and “consisting of” may bereplaced with either of the other two terms. The terms and expressionswhich have been employed are used as terms of description and not oflimitation, and there is no intention that use of such terms andexpressions imply excluding any equivalents of the features shown anddescribed in whole or in part thereof, but it is recognized that variousmodifications are possible within the scope of the invention claimed.Thus, it should be understood that although the present invention hasbeen specifically disclosed by preferred embodiments and optionalfeatures, modification and variation of the concepts herein disclosedmay be resorted to by those skilled in the art, and that suchmodifications and variations are considered to be within the scope ofthis invention as defined by the appended claims.

What is claimed is:
 1. A method of treating an auditory impairmentassociated with cochlear outer hair cell damage, dysfunction or loss ina subject comprising administering to said subject an effective amountof a composition comprising, as an active agent, one or more of acarboxy alkyl ester selected from the group consisting of: (a)3,4-O-dicaffeoylquinic acid, (b) 3,5-O-dicaffeoylquinic acid, (c)1,3-O-dicaffeoylquinic acid, (d) 4,5-O-dicaffeoylquinic acid, (e)1,5-O-dicaffeoylquinic acid, (f) 3-O-feruloylquinic acid, (g)4-O-feruloylquinic acid, (h) 5-O-feruloylquinic acid, (i)1-O-caffeoylquinic acid, (j) 3-O-caffeoylquinic acid, (k)4-O-caffeoylquinic acid, (l) 5-O-caffeoylquinic acid, (m)(1S,3R,4R,5R)-3-[3-(3,4-dihydroxyphenyl)-3R-hydroxypropanoyl]-1,4,5-trihydroxycyclohexanecarboxylicacid, (n)(1S,3R,4R,5R)-3-[3-(3,4-dihydroxyphenyl)-3S-hydroxypropanoyl]-1,4,5-trihydroxycyclohexanecarboxylicacid, (o)(1S,3R,4R,5R)-5-[3-(3,4-dihydroxyphenyl)-3R-hydroxypropanoyl]-1,3,4-trihydroxycyclohexanecarboxylicacid, (p)(1S,3R,4R,5R)-5-[3-(3,4-dihydroxyphenyl)-3S-hydroxypropanoyl]-1,3,4-trihydroxycyclohexanecarboxylicacid, (q)(1S,3R,4R,5R)-4-[3-(3,4-dihydroxyphenyl)-3R-hydroxypropanoyl]-1,3,5-trihydroxycyclohexanecarboxylicacid, (r)(1S,3R,4R,5R)-4-[3-(3,4-dihydroxyphenyl)-3S-hydroxypropanoyl]-1,3,5-trihydroxycyclohexanecarboxylicacid, (s) cis-5-O-caffeoylquinic acid, (t) 3-O-caffeoylquinic acidlactone, (u) 3-O-caffeoyl-4-O-feruloylquinic acid, and (v) apharmaceutically acceptable salt thereof, so as to restore cochlearouter hair cell function, thereby treating the auditory impairmentassociated with cochlear outer hair cell damage, dysfunction or loss inthe subject.
 2. The method of claim 1, wherein said composition furthercomprises an aqueous extract from Uncaria tomentosa bark.
 3. The methodof claim 1, wherein the composition is formulated for administrationselected from the group consisting of auricular, oral, parenteral,intraperitoneal, local, buccal, nasal, and topical administration. 4.The method of claim 1, wherein said composition is an aqueous extractfrom Uncaria tomentosa bark.
 5. The method of claim 1, wherein saidcomposition is in the form of a liquid, tablet or capsule.
 6. The methodof claim 1, wherein the auditory impairment is selected from the groupconsisting of permanent sensorineural hearing loss, tinnitus, loudnessrecruitment, hyperacusis, diplacusis and speech intelligibilitydeficits.
 7. The method of claim 1, wherein the active agent is lessthan or equal to 10,000 daltons.
 8. The method of claim 2, wherein theaqueous extract from Uncaria tomentosa bark comprises an active agent ofless than or equal to 10,000 daltons.
 9. The method of claim 1, whereincochlear outer hair cell function is sensory function as assessed byaudiological tests including distortion product otoacoustic emission(DPOAE).